Molecular conjugate

ABSTRACT

A method is disclosed for making a conjugate of two molecules using a hydrazide thiol linker. In a particular working embodiment, an Fc-specific antibody-enzyme conjugate is made using the method and demonstrated to provide exceptional staining sensitivity and specificity in immunohistochemical and in situ hybridization assays.

RELATED APPLICATION DATA

This patent application is a continuation of U.S. patent applicationSer. No. 12/687,564, filed on Jan. 14, 2010, now U.S. Pat. No.8,686,122, which is a continuation of U.S. patent application Ser. No.11/603,425, filed on Nov. 21, 2006, now abandoned, which claims thebenefit of U.S. Provisional Patent Application No. 60/739,794, filedNov. 23, 2005, all of which are incorporated herein by reference.

FIELD

The present invention relates to molecular conjugates, linkers formaking such conjugates, methods for making the conjugates and thelinkers, and methods of using the conjugates. More particularly, thepresent invention relates to Fc-specific antibody conjugates, hydrazidethiol linkers for preparing Fc-specific conjugates, methods for makingFc-specific conjugates, and methods of using Fc-specific antibodyconjugates.

BACKGROUND

A wide variety of methods have been developed for linking moleculestogether to form conjugates. Of particular interest are biomolecularconjugates that are typically prepared to combine the functionalities ofthe joined molecules into one construct. One type of biomolecularconjugate combines a biomolecule that specifically binds to anothermolecule (such as a nucleic acid, an antibody, a lectin or an avidin)and a detectable label (such as a fluorescent label, fluorescentnanoparticle or an enzyme).

Conjugates of antibodies and detectable labels (antibody conjugates) canbe used in immunoassays for detecting specific target molecules inbiological samples. The antibody portion of such conjugates specificallybinds to a target in the sample and the detectable label is utilized toprovide a detectable signal that indicates the presence/and or locationof the target. One type of conjugate that has become widely used,especially for immunohistochemical analysis, is a conjugate of anantibody and an enzyme (antibody-enzyme conjugate). A detectable signalis generated by adding a substrate to the sample under conditions wherethe enzyme portion of the antibody-enzyme conjugate converts thesubstrate to a detectable product (such as a colored, different-coloredor fluorescent product) at the site where the antibody portion is boundto its target.

Antibody conjugates are typically prepared using coupling reagents thatare characterized by having at least two reactive groups, one of whichis reacted with a functional group on the antibody and the other ofwhich is reacted with a functional group on the detectable label.However, coupling can lead to inactivation of either or both of theantibody and the detectable label. In particular, coupling candeactivate antibody-enzyme conjugates through steric effects or becausethe coupling reagents react with functional groups located on portionsof the antibody and/or enzyme that are critical for their specificityand/or catalytic activity. Furthermore, some coupling schemes lead toconjugates that have reduced water solubility.

Coupling schemes that can provide antibody-enzyme conjugates withreduced impairment of antibody specificity and/or enzyme activity aredesirable and enable greater sensitivities to be achieved inimmunochemical assays such as immunohistochemical assays. Greatersensitivity is of particular importance for automated processes whereadditional amplification steps are undesirable.

SUMMARY

A molecular conjugate that includes a hydrazide thiol linker isdisclosed. In one embodiment, an antibody-detectable label conjugate isprovided including a hydrazide thiol linker covalently bonded to the Fcportion of the antibody. The Fc-specific conjugate of this embodimentprovides improved detection sensitivity, thereby makingimmunohistochemical detection of a target molecule more amenable toautomation and high-throughput applications.

Also disclosed is a method for preparing a conjugate using a hydrazidethiol linker. In one embodiment, a protecting group for a thiol group ofthe linker is not needed because the linker is reacted with a firstmolecule under conditions where the thiol group is substantially presentin its neutral acid form and thus substantially unreactive. Under suchconditions, a covalent bond can be formed between a hydrazide group ofthe linker compound and a first molecule while substantially preservingthe thiol group for subsequent reaction with a thiol-reactive group of asecond molecule.

Hydrazide thiol linkers and methods for making hydrazide thiol linkersalso are disclosed. In addition, methods are described for using adisclosed conjugate to detect a target molecule in a sample such as atissue section or cytology sample. The methods of detecting a targetmolecule can be readily automated due to the improved sensitivityexhibited by the disclosed conjugates. In certain embodiments,multiplexed assays using the disclosed conjugates are provided, forexample, multiplexed assays employing disclosed antibody conjugateshaving fluorescent molecules or fluorescent nanoparticles as thedetectable label.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1D are a series of images showing staining patterns fordetection of Kappa in tonsil tissue using a disclosed Fc-specificantibody-alkaline phosphatase conjugate and using astreptavidin-alkaline phosphatase conjugate.

FIGS. 2A-2D are a series of images showing staining patterns fordetection of Lambda in tonsil tissue using a disclosed Fc-specificantibody-alkaline phosphatase conjugate and using astreptavidin-alkaline phosphatase conjugate.

FIGS. 3A-3D are a series of images showing staining patterns fordetection of CMV in lung tissue using a disclosed Fc-specificantibody-alkaline phosphatase conjugate and using astreptavidin-alkaline phosphatase conjugate.

FIGS. 4A-4D are a series of images showing staining patterns fordetection of EBER in spleen tissue using a disclosed Fc-specificantibody-alkaline phosphatase conjugate and using astreptavidin-alkaline phosphatase conjugate.

FIGS. 5A-5C are a series of images showing staining patterns fordetection of HPV in CaSki xenograft tissue using a disclosed Fc-specificantibody-alkaline phosphatase conjugate and using astreptavidin-alkaline phosphatase conjugate.

FIGS. 6A-6C are a series of images showing staining patterns fordetection of HPV in HeLa xenograft tissue using a disclosed Fc-specificantibody-alkaline phosphatase conjugate and using astreptavidin-alkaline phosphatase conjugate.

FIGS. 7A and 7B are images showing staining patterns for detection ofHPV in SiHa xenograft tissue using a disclosed Fc-specificantibody-alkaline phosphatase conjugate and using astreptavidin-alkaline phosphatase conjugate.

FIGS. 8A-8C are a series of images showing staining patterns fordetection of HPV in cytology samples using a disclosed Fc-specificantibody-alkaline phosphatase conjugate and using astreptavidin-alkaline phosphatase conjugate.

FIGS. 9A and 9B are images showing staining patterns for detection ofactin in muscle tissue using a disclosed Fc-specific antibody-alkalinephosphatase conjugate and using a streptavidin-alkaline phosphataseconjugate.

FIGS. 10A-10G are a series of images showing a comparison of thesensitivity of disclosed antibody-enzyme conjugates with each other andwith antibody-body enzyme conjugates prepared by other methods.

DETAILED DESCRIPTION OF SEVERAL ILLUSTRATIVE EMBODIMENTS

Further aspects of the invention are illustrated by the followingnon-limiting descriptions and examples, which proceed with respect tothe abbreviations and terms below.

I. Abbreviations

Ab—antibody

(Ab-AP)—antibody-alkaline phosphatase conjugate

AP—alkaline phosphatase

BSA—bovine serum albumin

CMV—cytomegalovirus

EBER—Epstein-Barr virus early RNA

DL—detectable label

Fc—fragment crystallizable

HRP—horseradish peroxidase

IHC—immunohistochemistry

ISH—in situ hybridization

MAL—maleimide

MBCH—mercaptobutyric acid carbohydrazide

MBH—mercaptobutyric acid hydrazide

NHS—N-hydroxy-succinimide

PEG—polyethylene glycol

SBM—specific binding molecule

II. Terms

The terms “a,” “an” and “the” include both singular and plural referentsunless the context clearly indicates otherwise.

The term “amination” as used herein refers to reaction of a carbonylgroup of an aldehyde or a ketone with an amine group, wherein anamine-containing compound such as an amine or a hydrazide reacts withthe aldehyde or ketone to first form a Shiff base that can thenreversibly rearrange to a more stable form, or optionally be reduced toprevent reversal of the reaction. “Reductive amination” conditionsinclude addition of a reducing agent, more typically addition of a mildreducing agent such as sodium cyanoborohydride or one of its co-geners,for example, sodium triacetoxyborohydride. Other mild reducing agentsthat can be employed include various amine boranes.

The term “antibody” collectively refers to an immunoglobulin orimmunoglobulin-like molecule (including IgA, IgD, IgE, IgG and IgM, andsimilar molecules produced during an immune response in any organism,for example, in mammals such as humans, goats, rabbits and mice), or afragment thereof, that specifically binds to a target (or a group ofhighly similar targets) to the substantial exclusion of binding to othermolecules. In some embodiments, an antibody specifically binds to atarget with a binding constant that is at least 10³ M⁻¹ greater, 10⁴ M⁻¹greater or 10⁵ M⁻¹ greater than a binding constant for other moleculesin a sample. In other embodiments, an antibody has a Kd value forbinding to an antigenic determinant (such as a hapten or epitope) thatis on the order of 10⁻⁶ M or lower, such as 10⁻⁹ M or lower, or even10⁻¹² M or lower. Kd values can, for example, be determined bycompetitive ELISA (enzyme-linked immunosorbent assay) or using asurface-plasmon resonance device such as the Biacore T100, which isavailable from Biacore, Inc., Piscataway, N.J. Antibody fragmentsinclude proteolytic antibody fragments [such as F(ab′)₂ fragments, Fab′fragments, Fab′-SH fragments and Fab fragments as are known in the art],recombinant antibody fragments (such as sFv fragments, dsFv fragments,bispecific sFv fragments, bispecific dsFv fragments, diabodies, andtriabodies as are known in the art), and camelid antibodies (see, forexample, U.S. Pat. Nos. 6,015,695; 6,005,079; 5,874,541; 5,840,526;5,800,988; and 5,759,808). Antibodies include both monoclonal andpolyclonal antibody preparations. Although an antibody of a disclosedconjugate can specifically bind any particular molecule or anyparticular group of highly similar molecules, in particular embodiments,the antibody comprises an anti-hapten antibody (which can, for example,be used to detect a hapten-labeled probe sequence directed to a nucleicacid sequence of interest). In particular embodiments, the antibodycomprises an anti-antibody antibody that can be used as a secondaryantibody in an immunoassay. For example, the antibody can comprise ananti-IgG antibody such as an anti-mouse IgG antibody, an anti-rabbit IgGantibody or an anti-goat IgG antibody.

The phrase “conditions where a thiol group of a hydrazide thiol linkeris substantially present in its neutral acid form” refers to conditions,such as conditions of pH, wherein less than about 1% of the thiol group(—SH; the protonated neutral acid form) of the linker is present in itsconjugate base form (—S⁻; unprotonated, negatively charged form). Forexample, under such conditions less than about 0.1%, less than about0.01%, or even less than about 0.001% of the linker can be in theconjugate base form. Conditions where the thiol group of the hydrazidethiol linker compound is substantially present in its neutral acid forminclude a pH of less than about 7, for example, a pH of less than about6 such as a pH of less than about 5.5. In particular embodiments, suchconditions include a range of pHs, for example, from a pH of about 3 toa pH of about 7, from a pH of about 4 to a pH of about 7, from a pH ofabout 4 to a pH of about 6, from a pH of about 4.5 to a pH of about 5.5,or any sub-range of each of these ranges. In other embodiments, theupper limit of the pH range in which a thiol group of a particularlinker is substantially present in its neutral acid form (less than 1%of the thiol group being present as the conjugate base form) can behigher than 7, such as a pH of 8. One of ordinary skill in the art canreadily determine an upper limit to the pH range in which a given thiolgroup will be substantially present in the neutral acid form using theHenderson-Hasselbach equation and a pKa value for a thiol group of thelinker. In yet other embodiments, a thiol group of a particular linkercan be substantially present in its neutral acid form in a solventsystem for which an accurate pH cannot be determined, and one ofordinary skill in the art will recognize that solvent systems that areless polar than water may help keep the thiol group in its neutral acidform at higher apparent pHs. Alternatively, an experimentaldetermination of whether under particular conditions a thiol group of alinker is substantially present in its neutral acid form can be made bydetermining whether the thiol will reduce a disulfide bond present inanother molecule. For example, a determination can be made of the numberof free thiol groups (for example, using Ellman's reagent) introducedinto a molecule having disulfides (such as an immunoglobulin) by contactwith the linker under the particular conditions of pH (or estimated pHfor non-aqueous systems). Addition of an excess of the hydrazide thiollinker (such as a 50-fold excess or more) over a period of time (such asan hour or more) can be followed by the determination of the averagenumber of free thiols introduced into the molecule. If free thiols aregenerated to a substantial degree (such as greater than an average oftwo thiols introduced per immunoglobulin molecule), it shows that thethiol of the linker is not substantially present in it neutral acid formunder the tested conditions. For example, at a pH of about 7, a onehundred-fold excess of the linker MBH relative to an immunoglobulin willproduce an average of about 2 thiols per immunoglobulin molecule. At alower pH of 5, a thousand-fold excess of the MBH linker will produce, onaverage, substantially less than 1 thiol per immunoglobulin in 24 hours.These results demonstrate that for the linker MBH, the thiol group issubstantially present in its neutral acid form at a pH of about 7 orlower, since as pH is lowered, the equilibrium between the neutral acidform and its conjugate base is shifted more towards the neutral acidform.

A “conjugate” refers to two or more molecules (and/or materials such asnanoparticles) that are covalently linked into a larger construct. Insome embodiments, a conjugate includes one or more biomolecules (such aspeptides, nucleic acids, proteins, enzymes, sugars, polysaccharides,lipids, glycoproteins, and lipoproteins) covalently linked to one ormore other molecules, such as one or more other biomolecules. In otherembodiments, a conjugate includes one or more specific-binding molecules(such as antibodies and nucleic acid sequences) covalently linked to oneor more detectable labels (such as fluorescent molecules, fluorescentnanoparticles, haptens, enzymes and combinations thereof).

A “detectable label” is a molecule or material that can produce adetectable (such as visually, electronically or otherwise) signal thatindicating the presence and/or concentration of the label in a sample.When conjugated to a specific binding molecule, the detectable label canbe used to locate and/or quantify the target to which the specificbinding molecule is directed. Thereby, the presence and/or concentrationof the target in a sample can be detected by detecting the signalproduced by the detectable label. A detectable label can be detecteddirectly or indirectly, and several different detectable labelsconjugated to different specific-binding molecules can be used incombination to detect one or more targets. For example, a firstdetectable label such as a hapten conjugated to a nucleic acid probe orantibody specific to a target can be detected indirectly through the useof a second detectable label that is conjugated to a molecule thatspecifically binds the first detectable label. Multiple detectablelabels that can be separately detected can be conjugated to differentspecific binding molecules that specifically bind different targets toprovide a multiplexed assay that can provide simultaneous detection ofthe multiple targets in a sample. A detectable signal can be generatedby any known or yet to be discovered mechanism including absorption,emission and/or scattering of a photon (including radio frequency,microwave frequency, infrared frequency, visible frequency andultra-violet frequency photons). Detectable labels include colored,fluorescent, phosphorescent and luminescent molecules and materials,catalysts (such as enzymes) that convert one substance into anothersubstance to provide a detectable difference (such as by converting acolorless substance into a colored substance or vice versa, or byproducing a precipitate or increasing sample turbidity), haptens thatcan be detected through antibody-hapten binding interactions usingadditional detectably labeled antibody conjugates, and paramagnetic andmagnetic molecules or materials. Particular examples of detectablelabels include enzymes such as horseradish peroxidase, alkalinephosphatase, acid phosphatase, glucose oxidase, β-galactosidase,β-glucuronidase or β-lactamase; fluorescent molecules such asfluoresceins, coumarins, BODIPY dyes, resorufins, and rhodamines (manyadditional examples of fluorescent molecules can be found in TheHandbook—A Guide to Fluorescent Probes and Labeling Technologies,Molecular Probes, Eugene, Oreg.); nanoparticles such as quantum dots(obtained, for example, from QuantumDot Corp, Invitrogen NanocrystalTechnologies, Hayward, Calif.; see also, U.S. Pat. Nos. 6,815,064,6,682596 and 6,649,138, each of which patents is incorporated byreference herein); metal chelates such as DOTA and DPTA chelates ofradioactive or paramagnetic metal ions like Gd³⁺; and liposomes, forexample, liposomes containing trapped fluorescent molecules. Where thedetectable label includes an enzyme, a detectable substrate such as achromogen, a fluorogenic compound, or a luminogenic compound can be usedin combination with the enzyme to generate a detectable signal (A widevariety of such compounds are commercially available, for example, fromInvitrogen Corporation, Eugene Oreg.). Particular examples ofchromogenic compounds include diaminobenzidine (DAB),4-nitrophenylphospate (pNPP), fast red, bromochloroindolyl phosphate(BCIP), nitro blue tetrazolium (NBT), BCIP/NBT, fast red, AP Orange, APblue, tetramethylbenzidine (TMB), 2,2′-azino-di-[3-ethylbenzothiazolinesulphonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN),nitrophenyl-β-D-galactopyranoside (ONPG), o-phenylenediamine (OPD),5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal),methylumbelliferyl-β-D-galactopyranoside (MU-Gal),p-nitrophenyl-α-D-galactopyranoside (PNP),5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazol (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blueand tetrazolium violet. Alternatively, an enzyme can be used in ametallographic detection scheme. Metallographic detection methodsinclude using an enzyme such as alkaline phosphatase in combination witha water-soluble metal ion and a redox-inactive substrate of the enzyme.The substrate is converted to a redox-active agent by the enzyme, andthe redox-active agent reduces the metal ion, causing it to form adetectable precipitate. (See, for example, co-pending U.S. patentapplication Ser. No. 11/015,646, filed Dec. 20, 2004, PCT PublicationNo. 2005/003777 and U.S. Patent Application Publication No.2004/0265922; each of which is incorporated by reference herein).Metallographic detection methods include using an oxido-reductase enzyme(such as horseradish peroxidase) along with a water soluble metal ion,an oxidizing agent and a reducing agent, again to for form a detectableprecipitate. (See, for example, U.S. Pat. No. 6,670,113, which isincorporated by reference herein). Haptens are small molecules that arespecifically bound by antibodies, although by themselves they will notelicit an immune response in an animal and must first be attached to alarger carrier molecule such as a protein or a poly-nucleic acid togenerate an immune response. Examples of haptens include di-nitrophenol,biotin, digoxigenin, and fluorescein. Additional examples of oxazole,pyrazole, thiazole, nitroaryl, benzofuran, triperpene, urea, thiourea,rotenoid, coumarin and cyclolignan haptens are disclosed in co-pendingU.S. Provisional Patent Application No., 60/856,133, filed Nov. 1, 2006,which is incorporated by reference herein.

The term “Fc-specific conjugate” as used herein refers to a conjugate ofan immunoglobulin (or fragment thereof) in which a second molecule (suchas a detectable label) is covalently bonded to the glycosylated portionof the immunoglobulin (or a fragment of an immunoglobulin that retainsthe glycosylated portion). The glycosylated portion of an immunoglobulinis found in the Fc-region, which is a region of an immunoglobulin thatis located on the heavy chains of the immunoglobulin at positionsoutside of the portion of the immunoglobulin that is responsible for thespecific binding activity of the immunoglobulin.

The term “hydrazide group” refers to a hydrazide group (—CO—NH—NH₂); acarbohydrazide group (—NH—NH—CO—NH—NH₂); a semicarbazide group(—NH—CO—NH—NH₂); a thiosemicarbazide group (—NH—CS—NH—NH₂); athiocarbazide group (—NH—NH—CS—NH—NH₂); a carbonic acid dihydrazinegroup (—NH—CO—NH—NH—CO—NH—NH₂) or a sulfur containing derivativethereof; or a hydrazine carboxylate group (—O—CO—NH—NH₂) or asulfur-containing derivative thereof.

The term “hydrazide-reactive group” refers to a group of atoms that canreact with and form a covalent bond to a hydrazide group. Aldehyde andketone groups are examples of hydrazide-reactive groups.Hydrazide-reactive groups can be an intrinsic part of a molecule or canbe introduced to a molecule. One method for introducing an aldehydegroup (a hydrazide-reactive group) into polysaccharides andglycoproteins (including antibodies) is by oxidation such asperiodate-mediated oxidation of vicinal diols. In addition, double bondsin unsaturated fatty acids and ceramides can be converted to diols byosmium tetroxide and then oxidized by periodate to aldehydes.Furthermore, N-terminal serine and threonine residues of peptides andproteins can be selectively oxidized by periodate to aldehyde groups,permitting selective modification of certain proteins such ascorticotrophin and β-lactamase. Modification of periodate-oxidizedantibodies does not typically inactivate the antibody. Varying theconcentration of sodium periodate during the oxidation reaction givessome specificity with regard to the types of sugar residues that aremodified. For example, sodium periodate at a concentration of 1 mM at 0°C. typically cleaves only at the adjacent hydroxyls between carbon atoms7, 8 and 9 of sialic acid residues. Oxidizing polysaccharides using 10mM or greater concentrations of sodium periodate results in oxidation ofsugar residues other than sialic acid, thereby creating many aldehydeson a given polysaccharide. A suitable general protocol is described byHermanson, “Bioconjugate Techniques,” Academic Press, San Diego, 1996,ISBN 0-12-342336-8, which is incorporated by reference herein. Anothermethod for introducing aldehydes into biomolecules is through the use ofspecific sugar oxidases, for example, galactose oxidase, which is anenzyme that oxidizes terminal galactose residues to aldehydes,particularly in glycoproteins. When galactose residues are penultimateto sialic acid residues, neuramidase can be used to remove the sialicacid residue and expose galactose as the terminal residue. A protocolfor using a combination of neuramidase and galactose oxidase to oxidizegalactose residues to provide a reactive aldehyde group is provided inHermanson, “Bioconjugate Techniques,” Academic Press, San Diego, 1996,ISBN 0-12-342336-8, which is incorporated by reference herein. Aldehydesalso can be introduced to a molecule by reacting an amine group of amolecule with an NHS-aldehyde such as succinimidyl p-formylbenzoate(SFB) or succinimidyl p-formylphenoxyacetate (SFPA) (Invitrogen Corp.,Eugene, Oreg.). Alternatively, bis-aldehyde compounds such asglutaraldehyde can be used to modify an amine group to provide analdehyde group. Again, suitable protocols are provided in Hermanson,“Bioconjugate Techniques,” Academic Press, San Diego, 1996, ISBN0-12-342336-8, which is incorporated by reference herein.

The term “hydrazide thiol linker” refers to a molecule including one ormore hydrazide groups and one or more thiol groups (—SH) joinedcovalently through one or more linking atoms. The hydrazide group(s) andthiol group(s) of a hydrazide thiol linker can be joined through one ormore of various groups of atoms including methylene groups (—CH₂—),branched alkylene groups, additional hydrazide groups, aromatic groups,heteroaromatic groups, alicyclic groups, polyalkylene glycol groups(such as ethylene oxide groups; —O—CH₂—CH₂—), amide groups (—CONH—),amine groups (—NH—), ether groups (—O—), and combinations thereof. A“PEG-based hydrazide thiol linker” refers to a linker including 1 ormore ethylene glycol groups as part of its structure. A “multifunctionalhydrazide thiol linker” refers to a branched linker having at least onehydrazide group, at least one thiol group, and at least one additionalreactive group, such as an additional hydrazide group, an additionalthiol group, or any other group useful for preparing molecularconjugates. In some embodiments, a PEG-based hydrazide thiol linkercomprises a discrete PEG (dPEG) linker, which can be made from dPEGstarting materials such as those disclosed in U.S. Patent ApplicationPublication No. 20060020134, and can be purchased from Quanta Biodesign(Powell, Ohio). Examples of additional reactive groups that can beincluded in a polyfunctional hydrazide-thiol linker include maleimidegroups and active esters, such as N-hydroxysuccinimide esters, andhydroxy groups (—OH). Additional examples of reactive groups can befound in Hermanson, “Bioconjugate Techniques,” Academic Press, SanDiego, 1996, ISBN 0-12-342336-8, which is incorporated by referenceherein.

The term “sample” refers to any liquid, semi-solid or solid substance(or material) in or on which a target can be present. In particular, asample can be a biological sample or a sample obtained from a biologicalmaterial. Examples of biological samples include tissue samples andcytology samples.

The term “specific binding molecule” refers to a molecule thatspecifically binds to a second molecule. “Specifically binds” means thatthe specific-binding molecule binds to the second molecule to thesubstantial exclusion of other molecules that are present in a sample(for example, the binding constant of the specific-binding molecule isat least 10² M⁻¹ greater, 10³ M⁻¹ greater, 10⁴ M⁻¹ greater or 10⁵ M⁻¹greater than a binding constant for other molecules in the sample).Examples of specific binding molecules include nucleic acids, receptors,antibodies, enzymes, lectins and avidins. Examples of specific-bindinginteractions in which specific binding molecules can participate includeformation of duplexes and triplexes of nucleic acid sequences,receptor-ligand interactions (such as folate-folate receptorinteractions), antibody-antigen interactions, enzyme-substrateinteractions, lectin-sugar reactions and avidin-biotin interactions(such as streptavidin-biotin interactions).

The term “target” refers to any molecule for which the presence,location and/or concentration is or can be determined. Examples oftarget molecules include proteins, nucleic acid sequences, and haptens,such as haptens covalently bonded to nucleic acid sequences or proteins.Target molecules are typically detected using one or more conjugates ofa specific binding molecule and a detectable label.

The term “thiol-reactive group” refers to an atom or atoms that canreact with and form a covalent bond with a thiol group. A thiol reactivegroup can be an intrinsic part of a molecule or can be introduced to themolecule through reaction with one or more other molecules. Examples ofthiol-reactive groups include non-polymerizable Michael acceptors,haloacetyl groups (such as bromoacetyl and iodoacetyl groups), alkylhalides, maleimides, aziridines, acryloyl groups, vinyl sulfones,benzoquinones, aromatic groups that can undergo nucleophilicsubstitution such as fluorobenzene groups (such as tetra andpentafluorobenzene groups), and disulfide groups such as pyridyldisulfide groups and thiols activated with Ellman's reagent. Additionalexamples of each of these types of groups will be apparent to thoseskilled in the art. Further examples and information regarding reactionconditions and methods for exchanging one type of reactive group foranother to add a thiol-reactive group are provided in Hermanson,“Bioconjugate Techniques,” Academic Press, San Diego, 1996, ISBN0-12-342336-8, which is incorporated by reference herein. In aparticular embodiment, a heterobifunctional linker molecule is attachedto a molecule to introduce a thiol-reactive group. For example, a linkerhaving a maleimide group and an N-Hyroxysuccinimide (NHS) group can beattached to an amine group on a molecule through the NHS group, therebyproviding the molecule with a thiol-reactive maleimide group that can bereacted with a thiol group on another molecule (such as one introducedusing a hydrazide thiol linker and the disclosed method) to form aconjugate.

III. Overview

One of ordinary skill in the art will recognize that the disclosedmethod can be used to join any combination of molecules havingfunctional groups that can react with a hydrazide thiol linker. Thenon-limiting description that follows focuses on antibody conjugates,and more particularly, on antibody-enzyme conjugates, but should not beconstrued as a limitation on the scope of the invention. Although thespecifically disclosed conjugates are antibody-enzyme conjugates,conjugates between other biomolecules (such as nucleic acid sequences)and other detectable labels (such as haptens, fluorescent labels,fluorescent nanoparticles and fluorescent proteins, such as greenfluorescent protein) are contemplated and fall within the scope of thedisclosure.

Accordingly, in one aspect, a method is disclosed for forming aconjugate of two or more molecules. The method includes reacting ahydrazide thiol linker with a first molecule (such as an antibody)having a hydrazide-reactive group (such as an aldehyde) to form athiolated first molecule. The reaction is carried out under conditionswhere a thiol group of the hydrazide thiol linker is substantiallypresent in its neutral acid (protonated) form. The thiolated firstmolecule can then be reacted with a second molecule having athiol-reactive group (such as a maleimide group introduced to the secondmolecule) to form the conjugate. In a particular embodiment, thereaction of the first molecule with the linker is carried out at a pHfrom about pH=4 to about pH=7. In other particular embodiments, thehydrazide thiol linker can be a PEG-based hydrazide thiol linker, amultifunctional hydrazide thiol linker, or a PEG-based multifunctionalhydrazide thiol linker.

In another embodiment, the method can be used to covalently join aspecific binding molecule to a detectable label. In a more particularembodiment, the method can be used to link a first molecule having aglycosylated portion to another molecule. In this embodiment, theglycosylated portion is first oxidized to generate an aldehyde groupthat can be reacted with a hydrazide thiol linker. In an even moreparticular embodiment, the glycosylated first molecule can be anantibody that has a glycosylated Fc region. An Fc-specific thiolatedantibody is formed by reaction with a hydrazide thiol linker, and theFc-specific thiolated antibody can be reacted with a detectable labelhaving a thiol-reactive group.

In another aspect, a variety of hydrazide thiol linkers and methods formaking the same are provided as outlined in the Synthetic Overview andspecific Examples that follow. A further aspect is a conjugate preparedwith a disclosed linker. In an additional aspect, a kit is disclosedthat includes a disclosed linker and instructions for performing thedisclosed method for making a conjugate. Also disclosed are methods forusing disclosed conjugates to detect a target in a sample.

IV. Synthetic Overview

A. Preparation of Hydrazide Thiol Linkers

Although any hydrazide thiol linker can be used in the disclosed methodof making a conjugate, in one embodiment, a hydrazide thiol linker canbe provided by reacting a thiolactone with hydrazine, carbohydrazide ora dihydrazide according to Scheme 1 below, wherein n=1, 2 or 3, R₁ is H,—CONHNH₂, or —CO-A-CONHNH₂, where A is a divalent group having between 1and 100 carbon atoms that can be interrupted by one or more heteroatoms(for example, O, N or S), and can be substituted, for example, with oneor more alkyl, hydroxyl, alkoxy, acyl, carboxy, halogen, sulfonate, oxo,phosphonate and/or amine groups. In more particular embodiments, A is adivalent group consisting of 1-10 methylene groups (—CH₂—) and/or 1-24ethylene oxide (—CH—CH₂—O—) groups. In even more particular embodiments,A is a divalent group consisting of 1-6 methylene groups or 4-12ethylene oxide groups.

A wide variety of hydrazide thiol linkers that can be used in thedisclosed method also can be provided according to scheme 2 below. Inthis scheme, Z is a divalent group having from 1 to 100 carbon atoms,wherein the divalent group can be interrupted by one or more heteroatoms(for example, O, N or S), and can be substituted, for example, with oneor more hydroxyl, alkoxy, acyl, carboxy, halogen, sulfonate, oxo,phosphonate and/or amine groups. In more particular embodiments, Z is adivalent group consisting of 1-10 methylene groups (—CH₂—) and/or 1-24ethylene oxide (—CH—CH₂—O—) groups. In even more particular embodiments,Z is a divalent group consisting of 1-6 methylene groups or 4-12ethylene oxide groups. R₂ is H, —CONHNH₂, or —CO-A-CONHNH₂, where A is adivalent group having between 1 and 100 carbon atoms that can beinterrupted by one or more heteroatoms (for example, 0, N or S), and canbe substituted, for example, with one or more alkyl, hydroxyl, alkoxy,acyl, carboxy, halogen, sulfonate, oxo, phosphonate and/or amine groups.

In some embodiments, a PEG-based hydrazide thiol linker that can be usedin the disclosed method is provided and is prepared according to scheme3. In scheme 3, m=2 to 50; R₃ is H, —CONHNH₂, or —CO-A-CONHNH₂, where Ais a divalent group having between 1 and 100 carbon atoms that can beinterrupted by one or more heteroatoms (for example, O, N or S), and canbe substituted, for example, with one or more alkyl, hydroxyl, alkoxy,acyl, carboxy, halogen, sulfonate, oxo, phosphonate and/or amine groups;and X and Y are independently a bond or a divalent group having 1 to 20carbon atoms. In more particular embodiments, A is a divalent groupconsisting of 1-10 methylene groups (—CH₂—) and/or 1-24 ethylene oxide(—CH—CH₂—O—) groups. In even more particular embodiments, A is adivalent group consisting of 1-6 methylene groups or 4-12 ethylene oxidegroups. The X and Y divalent groups can be interrupted by one or moreheteroatoms (for example, O, N or S), and can be substituted, forexample, with one or more alkyl, hydroxyl, alkoxy, acyl, carboxy,halogen, sulfonate, oxo, phosphonate and/or amine groups. In moreparticular embodiments, X and Y are independently a bond or —(CH₂)_(p)—where p=1 to 3. The carbodiimide used in the coupling reaction can beany carbodiimide that provides the desired coupling according to thescheme. Examples of suitable carbodiimides include DCC(N,N′-dicyclohexylcarbodiimide), and DIC (N,N′-diisopropylcarbodiimide).In a working embodiment that is discussed below, DCC is used toaccomplish the coupling.

In other embodiments, a multifunctional hydrazide thiol linker that canbe used in the disclosed method is provided. Schemes 4a, 4b, 4c, and 4dbelow show general methods for preparing multifunctional linkers fromhomocysteine, lysine, glutamic acid and homoserine, respectively. InSchemes 4a, 4b, 4c and 4d, D is a divalent group having from 1 to 100carbon atoms, wherein the divalent group can be interrupted by one ormore heteroatoms (for example, O, N or S), and can be substituted, forexample, with one or more alkyl, hydroxyl, alkoxy, acyl, carboxy,halogen, sulfonate, oxo, phosphonate and/or amine groups. In moreparticular embodiments, D is a divalent group consisting of 1-10methylene groups (—CH₂—) and/or 1-24 ethylene oxide (—CH—CH₂—O—) groups.In even more particular embodiments, D is a divalent group consisting of1-6 methylene groups or 4-12 ethylene oxide groups. Also in Schemes 4a,4b, 4c and 4d, R₄ is H, —CONHNH₂, or —CO-A-CONHNH₂, where A is adivalent group having between 1 and 100 carbon atoms that can beinterrupted by one or more heteroatoms (for example, O, N or S), and canbe substituted, for example, with one or more alkyl, hydroxyl, alkoxy,acyl, carboxy, halogen, sulfonate, oxo, phosphonate and/or amine groups.In more particular embodiments, A is a divalent group consisting of 1-10methylene groups (—CH₂—) and/or 1-24 ethylene oxide (—CH—CH₂—O—) groups.In even more particular embodiments, A is a divalent group consisting of1-6 methylene groups or 4-12 ethylene oxide groups.

In other particular embodiments, a PEG-based multifunctional hydrazidethiol linker that can be used in the disclosed method is provided.Schemes 5a, 5b and 5c illustrate general synthetic schemes that can beused to provide such linkers. In these schemes, p=2 to 50 and R₅ is H,—CONHNH₂, or —CO-A-CONHNH₂, where A is a divalent group having between 1and 100 carbon atoms that can be interrupted by one or more heteroatoms(for example, O, N or S), and can be substituted, for example, with oneor more alkyl, hydroxyl, alkoxy, acyl, carboxy, halogen, sulfonate, oxo,phosphonate and/or amine groups. In more particular embodiments, A is adivalent group consisting of 1-10 methylene groups (—CH₂—) and/or 1-24ethylene oxide (—CH—CH₂—O—) groups. In even more particular embodiments,A is a divalent group consisting of 1-6 methylene groups or 4-12ethylene oxide groups. R₇ can be H, alkyl or a protecting group.

B. Preparation of Fc-specific Antibody Conjugates

In one embodiment, a conjugate including a hydrazide thiol linkercomprises a conjugate of an antibody and a detectable label. In aparticular embodiment, the conjugate comprises an Fc-specific conjugateof an antibody and a detectable label. In a more particular embodiment,the conjugate comprises an Fc-specific conjugate of an antibody and anenzyme such as alkaline phosphatase. Scheme 6 illustrates a method ofadding a hydrazide thiol linker to an antibody in an Fc-specific manner.

In Scheme 6, an antibody having a glycosylated Fc-portion issite-specifically oxidized to generate one or more aldehyde groups inthe sugar moieties of the glycosylated Fc-portion. The aldehyde group(s)is (are) then reacted with a hydrazide thiol linker under conditionswhere the thiol group of the hydrazide thiol linker is substantiallyprotonated (substantially in its neutral acid form). Under suchconditions, the hydrazide group of the linker is covalently bonded tothe Fc-portion of the antibody while leaving the thiol groupsubstantially unreacted (such as substantially unreacted with disulfidelinkages in the antibody) and thus retained for later reaction with asecond molecule having a thiol-reactive group such as a detectable labelhaving a thiol-reactive group. The reaction desirably includes furtherreaction with a mild reductant (an example of a reductive amination) toform a more stable hydrazone. Coupling of the thiolated antibody with adetectable label having a thiol-reactive group (such as a maleimidegroup) is illustrated in Scheme 7.

V. EXAMPLES

The following non-limiting examples of working embodiments are providedto further illustrate certain aspects of the invention.

Example 1 Synthesis of Mercaptobutyric Acid Hydrazide (MBH)

In a particular working embodiment, a hydrazide thiol linker wasprepared from γ-butyrothiolactone according to Scheme 8.

In particular, to a stirred solution of hydrazine monohydrate (2.43 ml,50 mmol) was slowly added γ-butyrothiolactone (0.43 ml, 5 mmol). After 4hours the excess hydrazine was removed in vacuo. The crude product waspurified by flash chromatography (SiO₂, 1:19 MeOH/MeCN) to give thedesired product as a colorless oil. Yield: 599 mg (89%): ¹H NMR (250MHz, CDCl₃) δ 7.56 (s, 1 H), 3.89 (s, 2 H), 2.56-2.47 (q, J=6.9 Hz, 2H), 2.28-2.22 (t, J=7.0 Hz, 2H), 1.94-1.83 (p, J=7.0 Hz, 2 H), 1.35-1.29(t, J=8.0 Hz, 1 H); ¹³C NMR (62.9 MHz, CDCl₃) δ 173.02, 32.38, 29.16,23.94; ESI-HRMS m/z 135.05955 (M+H⁺, C₄H₁₁N₂OS calc'd 135.05921).

Example 2 Synthesis of Mercaptobutyric Acid Carbohydrazide (MBCH)

In another particular working embodiment, a carbohydrazide thiol linkerwas prepared from γ-butyrothiolactone according to Scheme 9.

In particular, γ-butyrothiolactone (0.43 ml, 5 mmol) was diluted inacetonitrile (5 ml) and then slowly added to a solution ofcarbohydrazide (2.25 g, 25 mmol) in deionized water (5 ml). The reactionwas stirred at 40° C. for 18 h, and then concentrated in vacuo. Thecrude product was removed by filtration with acetonitrile and flashchromatography (SiO₂, 1:19 MeCN/MeOH) to give the product as a whitesolid. Yield: 672 mg (70%): ¹H NMR (250 MHz, D₂O) δ 2.62-2.56 (t, J=7.1Hz, 2 H), 2.47-2.41 (t, J=7.4 Hz, 2 H), 1.98-1.87 (m, 2 H); ¹³C NMR(62.9 MHz, D₂O) δ 179.14, 163.94, 34.86, 31.74, 25.91; ESI-HRMS m/z215.05818 (M+Na⁺, C₅H₁₂N₄NaO₂S calc'd 215.25787).

Example 3 Synthesis of Mercapto-dPEG₄-hydrazide

In yet another particular working embodiment, a PEG-based hydrazidethiol linker was prepared according to Scheme 10 to provide amercapto-dPEG hydrazide.

Acetyl-S-dPEG₄™-NHS ester (Quanta Biodesign, Powell, Ohio; 580 mg, 1.38mmol) was slowly added to anhydrous hydrazine (10 ml), and was stirredfor 18 h at ambient temperature. The reaction was concentrated in vacuoto give the crude product. Flash chromatography (SiO₂, 199:1 MeCN/AcOH)gave the product as a colorless oil. Yield: 240 mg (59%): ¹H NMR (250MHz, CDCl₃) δ 8.04 (s, 1 H), 3.88 (s, 2 H), 3.68-3.52 (m, 17 H),2.65-2.60 (t, J=6.3 Hz, 2 H), 2.43-2.39 (t, J=5.8 Hz, 2H); ¹³C NMR (62.9MHz, CDCl₃) δ 171.94, 72.74, 70.52, 70.49, 70.38, 70.15, 70.09, 66.72,35.17, 24.12; ESI-HRMS m/z 319.13073 (M+Na⁺, C₁₁H₂₄N₂NaO₅S calc'd319.13036). An acetyl-S-dPEG₈™-NHS ester also is commercially availablefrom Quanta Biodesign, (Powell, Ohio). In general, amercapto-dPEG-hydrazide can have the formulaH₂N—NH—CO—(CH₂—CH₂—O)_(t)—CH₂—CH₂—SH, where t=2 to 50.

Example 4 Synthesis of Conjugates of IgG and Alkaline Phosphatase

An Fc-specifically thiolated immunoglobulin was prepared according toScheme 11.

Specifically, to a solution of polyclonal antibody (1.5 ml, 3.0 mg/ml)was added sodium periodate (0.5 ml, 10 mg/ml in deionized water) for afinal periodate concentration of 11.7 mM. The reaction solution wasrotated for 2 hours before being passed through a PD-10 desalting column(0.1 M NaOAc, 1 mM EDTA, pH=5.0) to remove excess periodate. A hydrazidethiol linker (MBH, AMBH, MBCH or Mercapto-dPEG₄-hydrazide) was added ina 1000-fold molar excess to the antibody followed by sodiumcyanoborohydride (3.14 mg, 50 μmol), and the reaction was rotated for aperiod of 18 h before being concentrated to a final volume of 1 ml. Sizeexclusion chromatography (Superdex 200; 0.1 M NaOAc, pH=5.0) gave thepurified thiolated antibody. The number of thiols was quantitatedthrough a modified Ellman's assay (see, for example, Hermanson,“Bioconjugate Techniques,” Academic Press, San Diego, 1996, ISBN0-12-342336-8, which is incorporated by reference herein). Thisprocedure yielded an average of 3-5 thiol groups per antibody.

Reaction of a hydrazide thiol linker with an aldehyde group introducedto the Fc region of an immunoglobulin is advantageously performed at amildly acidic pH, for example, a pH between 4 and 6 such as a pH near 5.Without wishing to be bound by theory, it is likely that at such mildlyacidic pHs the aldehyde group is electrophilically activated byprotonation of the aldehyde oxygen and, concurrently, the hydrazidegroup (pKa of about 4) is not substantially protonated and remainshighly nucleophilic, thereby facilitating the reaction between thealdehyde group and the hydrazide group. Since such mildly acidicconditions also represent conditions where the sulfur of the thiol groupis substantially protonated (substantially present in its neutral acidform) and thus unable to react with disulfides linking the heavy andlight chains of an immunoglobulin, the reaction is facile and yet lesslikely to disrupt immunoglobulin structure. Furthermore, a free thiolgroup is maintained for further reaction to form a conjugate.

Thiol-reactive maleimide groups were introduced to alkaline phosphataseaccording to Scheme 12.

Specifically, alkaline phosphatase (Biozyme, San Diego, Calif.), whichwas received in a reactive buffer containing Tris, was passed through aPD-10 column in order to exchange the AP into a non-reactive buffer (0.1M sodium phosphate, 0.1 M sodium chloride, 1 mM magnesium chloride, 0.1mM zinc chloride, pH=7.5). Then, to a solution of alkaline phosphatase(0.8 ml, 17.5 mg/ml) a 100-fold excess of NHS-dPEG₁₂-MAL (QuantaBiodesign, Powell, Ohio) was added and the reaction was rotated for aperiod of 1 h. Size exclusion chromatography (Superdex 200; 0.1 M Tris,1 mM MgCl₂, 0.1 mM ZnCl₂, pH=7.5) yielded the purifiedmaleimido-alkaline phosphatase. The number of maleimides was quantitatedusing a modified Ellman's assay (see, for example, Hermanson,“Bioconjugate Techniques,” Academic Press, San Diego, 1996, ISBN0-12-342336-8, which is incorporated by reference herein), and onaverage 17-25 maleimide groups were introduced to each alkalinephosphatase enzyme.

The final conjugation of the thiolated Ab and the reactive AP was thenperformed at a pH above 7, which in this instance allowed for fastformation of a conjugate by reaction of the thiol on the Ab (present toa greater extent in the conjugate base thiolate form at higher pHs) andthe thiol-reactive maleimide group introduced to alkaline phosphatase.Scheme 10 below depicts the final conjugation of the thiolated Ab andthe thiol-reactive AP.

Specifically, the purified maleimido-alkaline phosphatase was combinedwith the purified thiolated antibody in a 1:1 molar ratio and rotatedfor a period of 18 h. Size exclusion chromatography (Superdex 200; 0.1 MTris, 1 mM MgCl₂, 0.1 mM ZnCl₂, pH=7.5) gave the purified conjugatewhich was diluted to an A₂₈₀=0.0087 into a 1:1 dilution of Stabilzyme™AP enzyme-stabilizing diluent (SurModics, Eden Prairie, Minn.) andanalyzed on tissue as described in the examples that follow. Theresulting conjugates showed unprecedented staining sensitivities in avariety of tissues, as shown in the Examples that follow.

The synthesis of the Ab-AP conjugate according to this procedureproduces a 1:1 conjugate having a median molecular weight ofapproximately 270 kDa. This is true regardless of the antibody used toprepare the conjugate (such as goat anti-mouse IgG, goat anti-rabbit IgGand rabbit anti-DNP antibodies). The crude chromatograms, obtained afterconjugation show overlap between product and starting material (medianmolecular weight of 145 kDa), which can be taken into considerationduring the purification process.

Example 5 Detection of Kappa in Tonsil Tissue

In this example, the performance of an Ab-AP conjugate prepared usingMBH according to the procedure of Example 4 was evaluated for itsdetection sensitivity in an in-situ hybridization (ISH) assay. Theprocedure utilized was adapted from a standard ISH protocol available ona BenchMark® automated slide staining instrument (Ventana MedicalSystems, Inc., Tucson, Ariz.). The automated staining protocol was asfollows.

A paraffin-embedded tonsil tissue sample on a slide was heated to 75° C.for 4 minutes and treated twice with EZPrep™volume adjust (VentanaMedical Systems, Inc., Tucson, Ariz.) at 75° C. before application ofLiquidCoverslip™ (Ventana Medical Systems, Inc., Tucson, Ariz.) withEZPrep™ volume adjust. After 4 minutes at 75° C., the slide was rinsedand EZPrep™ volume adjust was added along with LiquidCoverslip™ tode-paraffinize the tissue at 76° C. for 4 minutes. Liquid coverslip wasdispensed to cover the EZ-Prep. The slide was then heated to 90° C. for4 minutes and rinsed before cooling to 37° C. ISH-Protease 1 (100 μl,Ventana Medical Systems, Inc., Tucson, Ariz.) was added, incubated for 2minutes, and then rinsed, followed by the addition of afluorescein-labeled kappa nucleic acid probe (100 μl, INFORM® Kappa,Ventana Medical Systems, Inc., Tucson, Ariz.). After a 4 minuteincubation, the slide was heated to 85° C. for 12 minutes, then cooledto 47° C. and incubated for a further 64 minutes. The slide was rinsedfour times before the addition of a mouse anti-fluorescein primaryantibody (100 μl, Ventana Medical Systems, Inc., Tucson, Ariz.), whichwas incubated for 20 minutes, and then rinsed twice. At this pointeither a secondary antibody (for further amplification) wasautomatically added or the Ab-AP conjugate was manually added or addedautomatically from a dispenser. For the slides that were amplified, arabbit anti-mouse antibody (100 μl, Ventana Medical Systems, Inc.,Tucson, Ariz.) was added, incubated for 8 minutes and then the slide wasrinsed twice. In either case, once the AP-Ab conjugate (goat anti-rabbitor rabbit anti-mouse IgG conjugate, for samples with and without thesecondary antibody, respectively; 100 μl) was applied to the slide, theslide was incubated for 16 minutes and the slide was rinsed twice.Application of iView™ Blue Enhance enhancer (100 μl, Ventana MedicalSystems, Inc., Tucson, Ariz.) was followed by incubation for 4 minutesand application of both iView™ Blue NBT (100 μl, Ventana MedicalSystems, Inc., Tucson, Ariz.) and iView™ Blue BCIP (100 μl, VentanaMedical Systems, Inc., Tucson, Ariz.). BCIP, which is a substrate ofalkaline phosphatase generates an insoluble dark blue/purpleprecipitate, and NBT enhances the color of the BCIP. The slide was thenincubated for a period of 32 minutes, rinsed twice, and Counterstain NFR(100 μl, Ventana Medical Systems, Inc., Tucson, Ariz.) was added. Afterincubation with the counterstain for 6 minutes, the slide was againrinsed and taken off of the instrument. The slide was treated with adetergent wash before dehydration with the series ethanol, acetone thenxylene. A coverslip was added to the slide and the slide was viewed andphotographed through a microscope. A negative control slide that was nottreated with the kappa probe also was prepared in a similar fashion.

For comparison, a reference tonsil tissue sample was stained using asimilar procedure employing a SA-AP conjugate for detection of the kappaprobe (the procedure included addition of the secondary antibody asabove followed by extra amplification steps, wherein automated additionof a biotinylated anti-IgG antibody was performed instead of applicationof the Ab-AP conjugate, which was then followed by addition of the SA-APconjugate) The use of biotin-labeled antibodies and SA-AP conjugates isan industry standard for detection in automated ISH staining methods andserved as a reference on which to determine the relative performance ofthe Ab-AP conjugate. A negative control slide that was not treated withthe kappa probe also was prepared in a similar fashion using SA-APdetection. Coverslips were added to the slides and the slides wereviewed and photographed at 40× through a brightfield microscope.

FIG. 1 is a set of photomicrographs comparing the desired staining andbackground staining observed for kappa ISH detection in tonsil tissueusing the antibody-alkaline phosphatase conjugate and the SA-APconjugate. In FIG. 1A the staining of kappa without amplificationafforded by the secondary antibody and using the disclosed antibodyconjugate is shown. FIG. 1B shows a negative control slide treated withthe conjugate. FIG. 1C shows the staining of kappa using the SA-AP, andFIG. 1D shows the negative control for the same. A comparison of FIGS.1A and 1C demonstrates more defined staining by the antibody conjugate(even though fewer amplification steps were used), and a comparison ofFIGS. 1B and 1D demonstrates the lower background provided by theantibody conjugate. These results illustrate the superiority of thenon-biotin detection scheme enabled by the antibody conjugate.

Example 6 Detection of Lambda in Tonsil Tissue

The automated staining process described in Example 5 (with theexceptions that the fluorescein-labeled nucleic acid probe used wasspecific for Lambda; INFORM® Lamdba, Ventana Medical Systems, Inc.,Tucson, Ariz.; and ISH Protease 1 was incubated for 4 minutes) was usedto assess the performance of an Ab-AP conjugate for detection of Lambdain tonsil tissue. The Ab-AP conjugate was used without a secondaryantibody amplification step and was prepared using MBH as described inExample 4. For comparison, a reference slide was prepared using theSA-AP conjugate detection scheme described in Example 4.

The results are presented in FIG. 2. Specifically, FIGS. 2A and 2B showthe staining pattern obtained using the Ab-AP conjugate with and without(negative control) the addition of the Lambda specific nucleic acidprobe, respectively. FIGS. 2C and 2D show the staining pattern obtainedusing the SA-AP conjugate with and without (negative control) additionof the Lambda probe, respectively. A comparison of FIGS. 2A and 2C showsthat the staining pattern obtained using the Ab-AP conjugate is at leastas intense as that seen with the SA-AP conjugate, despite the processused for the Ab-AP conjugate involving one less amplification step. Acomparison of FIGS. 2B and 2D demonstrates that there is much lessbackground staining (evidenced by darker overall staining of the tissue)by the Ab-AP conjugate. Again, these results demonstrate theadvantageous reduction of background seen when the disclosed Ab-APconjugate is employed.

Example 7 Detection of CMV in Lung Tissue

The automated staining process described in Example 5 (with theexceptions that the fluorescein-labeled nucleic acid probe used wasspecific for CMV; INFORM® CMV, Ventana Medical Systems, Inc., Tucson,Ariz.; and ISH Protease 1 was incubated for 4 minutes) was used toassess the performance of an Ab-AP conjugate for detection of CMV inlung tissue. The Ab-AP conjugate was used without a secondary antibodyamplification step and was prepared using MBH as described in Example 4.For comparison, a reference slide was prepared using the SA-AP conjugatedetection scheme described in Example 4.

The results are presented in FIG. 3. Specifically, FIG. 3A shows thestaining pattern obtained using the Ab-AP conjugate in the presence ofthe probe, FIG. 3B shows the staining pattern obtained using the Ab-APconjugate in the absence of the probe, FIG. 3C shows the stainingpattern obtained using the SA-AP conjugate in the presence of the probe,and FIG. 3D shows the staining pattern using the SA-AP conjugated in theabsence of the probe. A comparison of FIG. 3A and 3C shows that stainingwith the Ab-AP conjugate is more defined than and at least as intense(despite having one less amplification step) as the staining provided bythe SA-AP conjugate. Furthermore, less background staining is seen forthe Ab-AP conjugate. The reduction in background provided by the Ab-APconjugate also is evident from a comparison of FIGS. 3B and 3D.

Example 8 Detection of EBER in Spleen Tissue

The automated staining process described in Example 5 (with theexceptions that the fluorescein-labeled nucleic acid probe used wasspecific for EBER; INFORM® EBER, Ventana Medical Systems, Inc., Tucson,Ariz.; and ISH Protease 1 was incubated for 4 minutes) was used toassess the performance of an Ab-AP conjugate for detection of EBER inspleen tissue. The Ab-AP conjugate was used without a secondary antibodyamplification step and was prepared using MBH as described in Example 4.For comparison, a reference slide was prepared using the SA-AP conjugatedetection scheme described in Example 4.

The results are presented in FIG. 4. Specifically, FIG. 4A shows thestaining pattern obtained using the Ab-AP conjugate in the presence ofthe probe, FIG. 4B shows the staining pattern obtained using the Ab-APconjugate in the absence of the probe, FIG. 4C shows the stainingpattern obtained using the SA-AP conjugate in the presence of the probe,and FIG. 4D shows the staining pattern using the SA-AP conjugated in theabsence of the probe. A comparison of FIG. 4A and 4C shows that stainingwith the Ab-AP conjugate is more defined than and at least as intense(despite having one less amplification step) as the staining provided bythe SA-AP conjugate. Furthermore, less background staining is seen forthe Ab-AP conjugate. The reduction in background provided by the Ab-APconjugate also is evident from a comparison of FIGS. 3B and 3D

Example 9 Detection of HPV in Tissue Xenografts

In this example, the performance of an Ab-AP conjugate that was preparedusing MBH according to the procedure of Example 4 was assessed, in part,to determine if it provided enough sensitivity to permit a furtherreduction in the number of steps needed to detect HPV sequences by ISH.The results show that it is possible to achieve a reduction in thenumber of steps needed for detection, thereby making the disclosed Ab-APconjugate very useful for an automated process where a reduction in thenumber of steps leads to a significant reduction in processing time andconcomitantly, assay cost.

The three detection schemes presented below as Schemes 14-16, wereperformed in an automated or semi-automated fashion. In each of theseschemes, a DNP-labeled nucleic acid probe that specifically binds to atleast a portion of an HPV nucleic acid sequence is first added to thesample. The subsequent steps depicted in these schemes are steps used todetect the presence of the probe bound to HPV nucleic acid.

In Scheme 14, an anti-DNP antibody is first bound to the probe. Ananti-IgG antibody is then added (first amplification step). In a secondamplification step, a biotinylated anti-IgG antibody is added. An SA-APconjugate is added, which binds to the biotinylated antibody, andstaining is accomplished through addition of a chromogenic substratethat is acted upon by AP. In scheme 15, the second amplification step iseliminated, and an anti-IgG antibody conjugated to AP is added prior tostaining rather than an SA-AP conjugate. In scheme 16, bothamplification steps are eliminated and the DNP-labeled probe is directlydetected by an anti-DNP antibody conjugated to AP.

HPV detection in a variety of cell lines grown in xenografts in SCIDmice was performed according to the following procedure adapted from astandard ISH protocol for the BenchMark® automated staining instrument(Ventana Medical Systems, Inc, Tucson, Ariz.). Paraffin-embedded tissueon a slide was heated to 75° C. for 4 minutes and then treated twicewith EZPrep™ volume adjust (Ventana Medical Systems, Inc., Tucson,Ariz.) at 75° C. before application of Liquid Coverslip™ (VentanaMedical Systems, Inc., Tucson, Ariz.) with EZPrep™ volume adjust. After4 minutes at 75° C., the slide was rinsed and EZPrep™ volume adjust wasadded to de-paraffinize the tissue at 76° C. for 4 minutes. LiquidCoverslip was dispensed to cover the EZPrep™. Cell Conditioner #2, cellconditioning solution (Ventana Medical Systems, Inc., Tucson, Ariz.) wasadded, the slide warmed to 90° C., and incubated for 8 minutes. This wasfollowed by another application of Cell Conditioner #2 and yet anotherincubation at 90° C. for 12 minutes. The slide was rinsed with ReactionBuffer (Ventana Medical Systems, Inc., Tucson, Ariz.), cooled to 37° C.and ISH-Protease 3 (100 μl, Ventana Medical Systems, Inc., Tucson,Ariz.) was added. After an incubation of 4 minutes, the slide was rinsedthree times before the application of a hybridization buffer (iView™Plus HybReady™ Solution, 100 μl, Ventana Medical Systems, Inc., Tucson,Ariz.) and was incubated for 4 minutes. Addition of a DNP-labeled HPVnucleic acid probe (HPV HR Probe, 200 μl, Ventana Medical Systems, Inc.,Tucson, Ariz.) was followed by an incubation of 4 minutes at 37° C., 12minutes at 95° C. and 124 minutes at 52° C. The slide was then rinsedtwice and warmed to 72° C. This last step was repeated two more timesbefore cooling the slide down to 37° C., and then, depending upon thedetection scheme followed, such slides were treated in an automated orsemi-automated fashion in one of three ways.

In one instance, as depicted in Scheme 14, an iView™+Anti-DNP (100 μl,Ventana Medical Systems, Inc, Tucson, Ariz.) primary antibody wasapplied, and incubated for 20 minutes. The slide was then rinsed twicebefore the addition of the iView™+Amp (100 μl, Ventana Medical Systems,Inc, Tucson, Ariz.) secondary antibody. Incubation of the conjugateoccurred for 8 minutes before rinsing the slide. The addition ofiVIEW™+Biotin-Ig (100 μl, Ventana Medical Systems, Inc, Tucson, Ariz.)was followed by a 12 minute incubation and addition of the iVIEW™+SA-AP(100 μl, Ventana Medical Systems, Inc, Tucson, Ariz.). The slide wasrinsed three times before the application of the iView™+Enhancer (100 μlVMSI) which was followed by incubation for 4 minutes and application ofboth iView™+NBT (100 μl, Ventana Medical Systems, Inc, Tucson, Ariz.)and iView™+BCIP (100 μl, Ventana Medical Systems, Inc, Tucson, Ariz.).The slide was then incubated for a period of 24 minutes, rinsed threetimes, and Counterstain NFR (100 μl, Ventana Medical Systems, Inc,Tucson, Ariz.) was added. After incubation with the counterstain for 4minutes, and slide was rinsed three more times and taken off of theinstrument. The slide was treated with detergent wash before dehydrationwith a series of ethanol, acetone and then xylene. A coverslip wasapplied to the slide and then the slide was viewed and photographedthrough a brightfield microscope.

In another instance, as depicted in Scheme 15, a rabbit anti-DNP primaryantibody was added (iView™ Plus anti-DNP primary antibody, 100 μl,Ventana Medical Systems, Inc., Tucson, Ariz.). The primary antibody wasincubated for 20 minutes and the slide was then rinsed twice before themanual addition (this step can also be automated to make the procedurefully automated) of the anti-rabbit IgG antibody conjugated to alkalinephosphatase (100 μl). Incubation of the conjugate occurred for 16minutes before rinsing the slide four times. Application of the iView™Plus Enhancer (100 μl, Ventana Medical Systems, Inc., Tucson, Ariz.) wasfollowed by incubation for 4 minutes and application of both NBT andBCIP for color development (iView™ Plus NBT and iView™ Plus BCIP, 100μl, Ventana Medical Systems, Inc., Tucson, Ariz.). The slide was thenincubated for a period of 24 minutes, rinsed three times, andCounterstain NFR (100 μl, Ventana Medical Systems, Inc., Tucson, Ariz.)was added. After incubation with the counterstain for 4 minutes, andslide was rinsed three more times and taken off of the instrument. Theslide was treated with a detergent wash before dehydration with ethanol,acetone and xylene. Following application of a cover slip, the slide wasviewed through a microscope and photographed at 40× using a brightfieldmicroscope.

In yet another instance, as depicted in scheme 16, the slide was treateddirectly with an alkaline phosphatase rabbit anti-DNP conjugate (100μl). The slide was incubated for 20 minutes and was then rinsed twicebefore the application of the iView™+Enhancer (100 μl, Ventana MedicalSystems, Inc, Tucson, Ariz.). This was followed by incubation for 4minutes and simultaneous application of both iView™+NBT (100 μl, VentanaMedical Systems, Inc, Tucson, Ariz.) and iView™+BCIP (100 μl, VentanaMedical Systems, Inc, Tucson, Ariz.). The slide was then incubated for aperiod of 24 minutes, rinsed three times, and Counterstain NFR (100 μl,Ventana Medical Systems, Inc, Tucson, Ariz.) was added. After incubationwith the counterstain for 4 minutes, and slide was rinsed three moretimes and taken off of the instrument. The slide was treated to adetergent wash before serial dehydration with ethanol, acetone andxylene. A coverslip was added to slide and it was viewed andphotographed at 40× using a brightfield microscope.

FIGS. 5-7 show the results of HPV detection in three different xenografttissue types. In FIGS. 5A, 5B and 5C, the staining patterns for HPVdetection in CaSki xenograft tissue according to each of Schemes 14, 15and 16 are shown, respectively. In FIGS. 6A, 6B and 6C, the stainingpatterns for HPV detection in HeLa xenograft tissue according to each ofSchemes 14, 15 and 16 are shown, respectively. In FIGS. 7A and 7B, thestaining patterns for single copy HPV detection (indicated by arrows) inSiHa xenograft tissue according to each of Schemes 14 and 15 are shown,respectively.

A comparison of FIGS. 5A and 5B demonstrates that the staining intensityprovided by detection according to Scheme 15 is greater than thatprovided according to Scheme 14 even though Scheme 15 includes two feweramplification steps. FIG. 5C demonstrates that HPV detection can beaccomplished without amplification using direct detection (Scheme 16)with an Ab-AP conjugate prepared according to Example 4. A comparison ofFIGS. 6A and 6B also demonstrates that the staining intensity providedby detection according to Scheme 15 is greater than that providedaccording to Scheme 14, even though Scheme 15 includes two fewer stepsof amplification. FIG. 6C demonstrates that HPV detection can beaccomplished without amplification using direct detection (scheme 16)with an Ab-AP conjugate prepared according to Example 4. A comparison ofFIGS. 7A and 7B shows that even single copies of HPV nucleic acidsequences can be detected with the detection process of Scheme 15.Overall, the results demonstrate that the superior sensitivity exhibitedby a disclosed Fc-specific Ab-AP facilitates automated detection byreducing the number of steps needed to detect HPV in tissue samples. Thereduction of the number of steps between Schemes 14 and 15 can reducethe total automated staining process time by 15% (from 6.5 hrs to 5.5hrs). Further reductions in process time can be realized by use ofScheme 16.

While a DNP labeled probe and specific types of antibodies weredescribed in this example, one of ordinary skill in the art willappreciate that many other haptens (such as fluorescein, digoxigenin andbiotin) can be used to label nucleic acid sequences and that the use ofmultiple nucleic acid probes to different targets, each having adifferent hapten label, can be used to permit multiplexed detection(such as with different detection antibodies conjugated to differentfluorescent nanoparticles that emit light of various differentwavelengths). Furthermore, one of ordinary skill in the art willrecognize that antibodies of other types and from other species thanthose described, other detectable labels, and other reagents forgenerating a detectable signal can be used in similar assays to detectother targets.

Example 10 Detection of HPV in Liquid-Based Preparations

Slides for the liquid-based prep HPV assay were prepared using theThinPrep® 2000 System slide preparation system (Cytyc Corporation,Marlborough, Mass.). Cells obtained through vaginal scraping are placedwithin a methanol-based, buffered preservative solution (ThinPrep®PreservCyt Solution, Cytyc Corporation, Marlborough, Mass.) and thenlayered onto the glass slide by the instrument.

The following is an adapted procedure from the Ventana BenchMark®Instrument: the liquid based prep slide was heated to 65° C. for 12minutes followed by an additional 4 minutes at 75° C. and rinsed twicewith Reaction buffer (Ventana Medical Systems, Inc, Tucson, Ariz.; 1.2ml) at 75° C. before application of the liquid cover slip (VentanaMedical Systems, Inc, Tucson, Ariz.). The slide was then rinsed with 0.9ml of Rinse Buffer (Ventana Medical Systems, Inc, Tucson, Ariz.)followed by the application of Cell Conditioner #2 cell conditioningsolution (Ventana Medical Systems, Inc, Tucson, Ariz.) and the slide waswarmed to 90° C. and incubated for 16 minutes. The slide was rinsed withReaction Buffer, cooled to 37° C. and ISH-Protease 3 (100 μl, VentanaMedical Systems, Inc, Tucson, Ariz.) was added. After an incubation of 4minutes, the slide was rinsed three times before the application ofiView™+HybReady (100 μl, Ventana Medical Systems, Inc, Tucson, Ariz.)which was incubated for 4 minutes. Addition of HPV HR Probe (200 μl,Ventana Medical Systems, Inc, Tucson, Ariz.) was followed by anincubation of 4 minutes at 37° C., 12 minutes at 95° C. and 124 minutesat 52° C. The slide was then rinsed twice and warmed to 72° C. This laststep was repeated two more times before cooling the slide down to 37° C.and adding iView™+Anti-DNP (100 μl, Ventana Medical Systems, Inc,Tucson, Ariz.).

For standard SA-AP detection (according to Scheme 14 above), the primaryantibody was incubated for 20 minutes and the slide was then rinsedtwice before the addition of the i™VIEW+Amp secondary antibody (VentanaMedical Systems, Inc, Tucson, Ariz., 100 μl). Incubation of the antibodyoccurred for 8 minutes before rinsing. Then, the i™VIEW+Biotin-IgGantibody conjugate (Ventana Medical Systems, Inc, Tucson, Ariz., 100 μl)was added followed by a 12 minute incubation and rinse step. Lastly, theiVIEW™+SA-AP conjugate (Ventana Medical Systems, Inc, Tucson, Ariz., 100μl) was added and after an 8 minute incubation, the slide was rinsedthree times with Reaction Buffer. For detection using the Ab-APconjugate as the secondary antibody (according to Scheme 15 above), theprimary antibody was incubated for 20 minutes and the slide was thenrinsed twice before the addition of the AP-IgG conjugate (100 μl).Incubation of the conjugate occurred for 8 minutes before rinsing threetimes with Reaction Buffer. For direct detection of the labeled probeusing the Ab-AP conjugate, the conjugate was incubated for 20 minutesbefore the slide was rinsed three times with Reaction Buffer.

In all three cases, the steps above were followed by application ofiVIEW+Enhancer (100 μl, Ventana Medical Systems, Inc, Tucson, Ariz.) wasfollowed by incubation for 4 minutes and application of both iVIEW™+NBT(100 μl, Ventana Medical Systems, Inc, Tucson, Ariz.) and iVIEW™+BCIP(100 μl, Ventana Medical Systems, Inc, Tucson, Ariz.). The slide wasthen incubated for a period of 24 minutes, rinsed three times, andCounterstain NFR (100 μl, Ventana Medical Systems, Inc, Tucson, Ariz.)was added. After incubation with the counterstain for 4 minutes, andslide was rinsed three more times and taken off of the instrument. Theslide was treated with a detergent wash before dehydration with ethanol,acetone and xylene and subsequent application of a cover slip to theslide, after which the slide was viewed through a microscope.

A comparison of FIGS. 8A and 8B shows that detection according to Scheme15 (see, Example 9), using an Ab-AP conjugate that was prepared usingMBH according to the procedure of Example 4, provides more intensestaining than provided by detection using an SA-AP conjugate accordingto Scheme 14 (see, Example 9). A comparison of FIGS. 8B and 8Cdemonstrates that direct detection using an anti-DNP Ab-AP conjugateaccording to Scheme 16 (see, Example 9) provides a signal that iscomparable to the signal provide by an SA-AP conjugate according toScheme 14. These results again demonstrate that the detectionsensitivity provided by an Fc-specific Ab-AP conjugate according toExample 4 permits a reduction in the number of steps needed to provideadequate signals, thereby facilitating automation.

Example 11 Detection of Actin in Muscle Tissue

In this example, immunohistochemical detection of a protein target(actin) using an Ab-AP conjugate prepared as described in Example 4 withan MBH linker was compared to the performance of a SA-AP conjugate.

The following is the adapted procedure from the Ventana BenchMark®Instrument: the paraffin coated tissue on the slide was heated to 75° C.for 4 minutes and treated twice with EZPrep™ volume adjust (VentanaMedical Systems, Inc, Tucson, Ariz.) at 75° C. before application of theliquid cover slip (Ventana Medical Systems, Inc, Tucson, Ariz.) withEZPrep™ volume adjust. After another 4 minutes at 76° C., the slide wasrinsed and Depar volume adjust (Ventana Medical Systems, Inc, Tucson,Ariz.) was added along with liquid cover slip to de-paraffinize thetissue. The slide was then cooled to 42° C. for 2 minutes, beforereaching the final temperature of 37° C. The primary antibody was thenadded (100 μl anti-muscle actin, Ventana Medical Systems, Inc, Tucson,Ariz.) and the slide incubated at 37° C. for 16 minutes. The slide wasthen rinsed twice and the alkaline phosphatase conjugated goatanti-mouse material (100 μl) was added and incubated 37° C. for 16minutes. The slide was rinsed once before the simultaneous addition ofEnhanced V-Red Enhancer (100 μl, Ventana Medical Systems, Inc, Tucson,Ariz.) and Enhance Naphthol (100 μl, Ventana Medical Systems, Inc,Tucson, Ariz.), and the slide was again incubated at 37° C. for 4minutes. This was followed by the addition of Enhance Fast Red A (100μl, Ventana Medical Systems, Inc, Tucson, Ariz.) an 8 minute incubationand the addition of Enhance Fast Red B (100 μl, Ventana Medical Systems,Inc, Tucson, Ariz.) with a final 8 minute incubation. After developmentof the stain, the slide was treated with a detergent wash beforedehydration with ethanol, acetone and xylene and subsequent applicationof a cover slip to the slide, after which the slide was viewed through amicroscope.

The results are presented in FIG. 9. Specifically, FIG. 9A shows thatdetection using the Ab-AP conjugate and a single amplification step issuperior to detection using an SA-AP conjugate and two amplificationsteps (FIG. 9B). These results again demonstrate the superior detectionsensitivity provided by Fc-specific antibody conjugates according to thedisclosure.

Example 12 Variation of Antibody Linker Length and Type

In this example, the effect of linker length and type on conjugatecomposition and staining characteristics was determined. Severalconjugates were prepared according to the method of Example 4, but usinga variety of hydrazide thiol linkers, specifically, conjugates preparedusing a thio-PEG₄-hydrazide linker, a mercaptobutyric acid hydrazide(MBH) linker, and a mercaptobutyric acid carbohydrazide (MBCH) linker.These conjugates were compared to each other and to a conjugate preparedthrough generation of thiols by reduction of immunoglobulin disulfides,specifically an Ab-AP conjugate prepared by the method described inco-pending U.S. Provisional Patent Application No. 60/675,759 thatinvolves generation of thiols via DTT reduction followed by conjugationsusing a PEG-based maleimide-NHS bifunctional linker. Also forcomparison, a commercially available acetamidomercaptobutyric acidhydrazide (AMBH, Invitrogen, Eugene, Oreg.) linker was used in themethod of Example 4 to generate an Ab-AP conjugate. In addition, anAb-AP conjugate prepared with a maleimido-hydrazide (EMCH;N[ε-Maleimidocaproic acid]hydrazide, Pierce Biotechnology, Rockford,Ill.) using the manufacturer's instructions was prepared and used in thestaining protocol for comparison. Furthermore, the Fc-specificconjugation method described in U.S. Pat. No. 5,191,066 employingcystamine was used to provide Fc-specific Ab-AP conjugate forcomparison.

Ellman's Assay results showed that between 3-5 thiols/Ab were added toan immunoglobulin through addition with the MBH and PEG-based hydrazidethiol linkers, 5-7 thiols/Ab for the AMBH and MBCH linkers, and 8-12thiols/Ab for the DTT reduction method. After coupling of the thiolsintroduced or generated in the immunoglobulin to maleimide-derivatizedAP, size exclusion chromatograms were obtained.

Size exclusion chromatograms were obtained using an AKTA Purifier LC (GEBiosciences, Uppsala, Sweden) using a Superdex 10/300 200 GL column and0.1 M Tris, 1 mM MgCl₂, 0.1 mM ZnCl₂, pH=7.5 as the mobile phase. Theflow rate was held at 1 ml/min in all cases. From the size exclusionchromatograms, it was determined that the best yield of conjugate wasobtained using AMBH. However, it began to precipitate out of solutionwhen stored at 2-8° C. for 48 hours. The other linkers all yieldedconjugates having similar size exclusion profiles.

FIG. 10 compares the staining as outlined in Example 6 of Kappa ontonsil tissue using the conjugates as the secondary antibody. FIG. 10Ashows the staining pattern seen for an Ab-AP conjugate prepared withEMCH. FIG. 10B shows the staining pattern seen for the Fc-specificcystamine method of U.S. Pat. No. 5,191,066. FIG. 10C shows the stainingpattern seen for the Ab-AP conjugate prepared by a DTT reduction methodaccording U.S. patent application Ser. No. 11/413,418, filed Apr. 27,2006. that utilized a dPEG-based bifunctional linker FIG. 10D shows thestaining pattern seen for the Ab-AP conjugated prepared with thecommercially available AMBH linker using the disclosed method ofFc-specific conjugation. FIG. 10E shows the staining pattern seen forthe Ab-AP conjugate prepared according to the disclosed Fc-specificconjugation method employing the disclosed MBH linker of Example 1. FIG.10F shows the staining pattern seen for the Ab-AP conjugate preparedaccording to the disclosed Fc-specific conjugation method employing thedisclosed dPEG₄ hydrazide thiol linker of Example 3. FIG. 10G shows thestaining pattern seen for the Ab-AP conjugate prepared according to thedisclosed Fc-specific conjugation method employing the disclosed MBCHhydrazide thiol linker of Example 2. A comparison of the stainingpatterns reveals the following trend for the staining intensity providedby the conjugates:EMCH<Cystamine<AMBH<MBCH<PEG4=DTT<MBHThe images illustrate the superior sensitivity that can be achieved byFc-specific conjugation of enzymes using the disclosed method andvarious disclosed and commercially available hydrazide thiol linkers.The disclosed method also yields superior conjugates to the cystamineFc-specific method and coupling with EMCH. Only the DTT-mediated methodof conjugation provides conjugates that give similar specificity andsensitivity.

Example 13 Variation of MBH Linker Excess

In this example, the dependence of conjugate composition and stainingcharacteristics on the excess of hydrazide thiol linker was determined.Synthesis of AP-IgG conjugates with MBH linker was carried out followingthe procedure of Example 4, however the molar excess of the MBH linkerwas varied from a five thousand-fold excess to a fifty-fold excess. Theresults from the Ellman's Assay showed the following number ofthiols/Ab: 5000×—9-15; 1000×—7-10; 500×—3-5; 100×—2-4; 50×—1-3. Analysisof the conjugates (5000×, 1000×, 500×, 100×, and 50×) after reactionwith the maleimide-derivatized Ab was performed by size exclusionchromatography and showed that the conjugates synthesized using a largerexcess of linker had a higher overall yield. However, the tissuestaining (anti-mouse—muscle, muscle actin; anti-rabbit—skin, S100) foreach of these conjugates showed that the 500× had the most intense stainwith the lowest amount of background.

Example 14 Variation of Alkaline Phosphatase Linker Length/Type

In this example, the dependence of conjugate composition and stainingcharacteristics on the length and type of linker used to addthiol-reactive groups to alkaline phosphatase was determined. Synthesisof AP-IgG conjugates with MBH linker was carried out following theprocedure of Example 4, but the following linkers were used to activatealkaline phosphatase for reaction with the thiolated antibody: LC-SMCC(Pierce, Rockford, Ill.), MAL-dPEG₈-NHS ester (Quanta Biodesign, Powell,Ohio), MAL-dPEG₄-NHS ester (Quanta Biodesign, Powell, Ohio) andMAL-dPEG₁₂-NHS ester (Quanta Biodesign, Powell, Ohio). Each of theselinkers was reacted with AP in a hundred-fold excess, in a buffer system(0.1 M sodium phosphate. 0.1 M NaCl, 1 mM MgCl₂, 0.1 mM ZnCl₂, pH=7.5)for 1 hour. The LC-SMCC had to be dissolved in dimethylformamide (DMF)and added to the AP, but not exceeding 10% total volume of DMF inbuffer. Ellman's Assay showed a maleimide incorporation of 20/AP for thePEG₁₂ and LC-SMCC linkers, 27/AP for the PEG₈ linker and 30/AP for thePEG₄ linker. After coupling to an Fc-thiolated antibody (made with MBH),size exclusion chromatograms were obtained upon purification. The PEG₁₂linker gave the highest conjugate yield, followed by the PEG₈, LC-SMCC,and the PEG₄ linkers. The tissue staining (anti-mouse—muscle, muscleactin; anti-rabbit—skin, S100) mirrored the conjugate yield with thePEG₁₂ conjugate giving the most intense staining.

Example 15 Variation of NHS-PEG₁₂-MAL Linker Excess

In this example, the dependence of conjugate composition and stainingcharacteristics on excess of NHS-PEG₁₂-MAL linker used to addthiol-reactive groups to alkaline phosphatase was determined. Synthesesof an AP-IgG conjugates according to the method of Example 4 wasperformed where the molar excess of a MAL-dPEG₁₂-NHS ester linker wasvaried from a five hundred-fold excess to a twenty-five-fold excess.

Ellman's Assay results showed maleimide incorporation of: 500×—34maleimides; 250×—29 maleimides; 100×—18-20 maleimides; 50×—17maleimides; 25×—15 maleimides. Analysis of the conjugates (500×, 250×,100×, 50×, and 25×), after reaction with the Fc-thiolated Ab, using sizeexclusion chromatography showed that the conjugates synthesized using alarger excess of linker had a larger yield and a higher percentage ofmaleimide incorporation. Tissue staining (anti-mouse—muscle, muscleactin; anti-rabbit—skin, S100) for each of the conjugates showed thatuse of 100× maleimide gave the sharpest, most intense staining.

Example 16 Variation of AP/Ab Molar Ratios

In this example, the dependence of conjugate composition and stainingcharacteristics on the ratio of the thiolated antibody (prepared with anMBH-linker) to the maleimide-derivatized AP (NHS-PEG₁₂-MAL linker) inthe final reaction was determined. The following ratios (Antibody/AP)were used: 2:1, 1:1, 1:2, and 1:3. The profiles of size exclusionchromatographs showed that maximum yield was obtained when the molarratio was 2 AP:1 Ab. However, the tissue staining of the conjugates(anti-mouse—muscle, muscle actin; anti-rabbit—skin, S100) demonstratedthat the best signal-to-noise ratio was seen with the 1:1 conjugate.

Example 17 Synthesis of Cross-linked AP

Alkaline phosphatase is a dimeric protein, and its stability can beincreased by cross-linking the enzyme to help prevent dissociation ofthe dimer. Alkaline phosphatase was cross-linked using the followingprocedure. Alkaline phosphatase (Biozyme, San Diego, Calif.; 17.5 mg,0.125 μmol) was exchanged into a different buffer from that in which itwas received (0.1 M sodium phosphate, 0.1 M sodium chloride, 1.0 mMmagnesium chloride, 0.1 mM zinc chloride, pH=7.5) and added toreconstituted, pre-oxidized, aldehyde-activated dextran (Avg. MolecularWt. 40,000; Pierce Biotechnologies, Rockford, Ill.; 5 mg, 0.125 μmol) inthe presence of sodium cyanoborohydride (1.6 mg, 25 μmol). The reactionmixture was then rotated for a period of one hour at room temperature.Excess aldehydes were quenched by ethanolamine (151 μl, 2.5 mmol)followed by addition of more sodium cyanoborohydride (157.1 mg, 2.5mmol). The reaction mixture was rotated for an additional one hour. Thecross-linked AP was isolated by size exclusion chromatography using anAkta Purifier (GE Biosciences, Uppsala, Sweden) equipped with a Superdex200 GL 10/300 column (GE Biosciences, Uppsala, Sweden). The flow ratewas 1 ml/min and the aqueous mobile phase was 0.1 M sodium phosphate,0.1 M sodium chloride, 1.0 mM magnesium chloride, 0.1 mM zinc chloride,at pH=7.5. The number of amines remaining after the reaction wasquantitated using a fluoraldehyde assay (Protein Assay TechnicalHandbook, Pierce Biotechnology, Rockford, Ill.), and on average 8-12amines remained following cross-linking. The cross-linked AP wasattached to MAL-dPEG₁₂-NHS ester (which reacted with the remainingamines) and was conjugated to an Fc-thiolated antibody as described inExample 4 to generate a conjugate including a cross-linked AP enzyme.Stability studies showed that cross-linking improved the stability ofthe conjugate in an avidin-containing diluent (Ventana Medical Systems,Inc, Tucson, Ariz.; P/N 95130). Specifically, at 45° C., the total lossof staining intensity on the 3^(rd) day for the conjugate with thecross-linked AP was 50%, whereas in the same diluent and at thetemperature, a conjugate prepared with a non-crosslinked AP lost 95% ofits staining intensity on the 1^(st) day.

Alternative methods for cross-linking AP to increase its stability areprovided in Bieniarz et al., Bioconj. Chem., 9: 390-398, 1998, Bieniarzet al., Bioconj. Chem., 9: 399-402, 1998, and U.S. Pat. No. 5,789,219.These methods also can be used to cross-link alkaline phosphataseenzymes for use in a disclosed conjugate.

Example 18 Analytical SDS PAGE of Alkaline Phosphatase Conjugates

In this example, the Fc-specificity of the conjugation method of Example4 is demonstrated by polyacrylamide gel electrophoresis under denaturingconditions. Six different preparations of the conjugate, 3 prepared withan anti-mouse IgG antibody and 3 prepared with an anti-rabbit IgGantibody were analyzed. Briefly, five to 20 μl of a 100-200 ng per μlsolution of each conjugate was mixed with 4×LDS gel loading buffer(Invitrogen, Carlsbad, Calif.), and 2-Mercaptoethanol was added to afinal concentration of 1 mM. The sample mixture was moderately heated at48-50° C. for 5 minutes. This temperature was chosen to minimizedissociation of the covalent linkage between the enzyme and theantibody, while still permitting dissociating the light and heavy chainsof the antibody portion of the conjugate by the 2-Mercaptoethanol. Eachsample was then cooled and added to different wells of a polyacrylamidegel (either a 1.0-mm-thick, pre-formed NuPAGE™ 4-20% polyacrylamide BisTris gel or a NuPAGE™ 3-8% polyacrylamide Tris acetate gel fromInvitrogen, Carlsbad, Calif.). The molecular weight standards used werepre-stained Multimark™ and Mark12 wide Range™ standards, both of whichwere purchased from Invitrogen (Carlsbad, Calif.). Electrophoresis wascarried out at 70 mA for 60 to 90 minutes at room temperature using aNovex XCell II cassette system (Invitrogen, Carlsbad, Calif.). Therunning buffer was MES-SDS or Tris Acetate-SDS buffer, for the 3-8% and4-20% gels, respectively. Gels were removed from the cassettes andwashed twice in deionized water for 5 minutes in order to remove the SDSand buffer. The SDS-PAGE gels were then fixed in ethanol/water/aceticacid [40:50:10 (v:v:v)] for 1 hour at room temperature and stained withCoomassie Blue R-250 dissolved in methanol/water/acetic acid [50:40:10(v:v:v), Sigma-Aldrich, St. Louis, Mo.]. The gels were stained for aminimum of 2 hours to a maximum of overnight by gentle rocking at roomtemperature. De-staining was carried out in the same manner as staining.The de-staining solution was identical to the staining solution minusthe dye. Gels were dried using an Invitrogen gel drying kit (Invitrogen,Carlsbad, Calif.). Analysis of the gels clearly showed for each of theconjugates the presence of a band at a molecular weight corresponding tothe light chain of the antibody. Also, for each conjugate, there was asubstantial absence of bands corresponding to the molecular weight ofthe heavy chain and of alkaline phosphatase. Instead, a series of bandsat higher molecular weights showed that the alkaline phosphatase wasselectively bound to the heavy chain of the IgGs for each conjugate.Since the heavy chain of an immunoglobulin includes the Fc region, theresults showed the Fc-site specific nature of the conjugation.

Example 19 Synthesis of an Fc-Specific Antibody-HRP Conjugate

In this example, preparation of an Fc-specific antibody conjugateincluding a PEG-based hydrazide thiol linker is described.Thiol-reactive maleimide groups were added to horseradish peroxidase asfollows. To a 4 mL amber vial was added 7.8 mg (15.2 μmol, 100 eq.) ofMAL-dPEG₄™NHS ester (Quanta Biodesign, Powell, Ohio), followed byhorseradish peroxidase (HRP; Pierce Biotechnology, Rockford, Ill.; 0.25ml, 25 mg/ml in 0.1 M Na₃PO₄, 0.15 M NaCl, pH=7.5). The vial was rotatedin the dark at ambient temperature for 1 hour before being purified bysize exclusion chromatography using an Akta Purifier equipped with aSuperdex 200 column (GE Biosciences, Uppsala, Sweden) using an aqueousbuffer solution (0.1 M Na₃PO₄, 0.15 M NaCl, pH=7.5). HRP containingfractions were pooled to give a solution of HRP-PEG₄-maleimide. The HRPconcentration was determined from the A₂₈₀ of the solution (ε₂₈₀=0.652ml cm⁻¹ mg⁻¹) and the number of maleimides was quantitated through amodified Ellman's assay to be between 6 and 8 maleimides per enzyme.

The purified maleimido-horseradish peroxidase was combined with apurified thiolated antibody (according to Example 4, prepared using anMBH linker) in a 3:1 molar ratio and rotated for a period of 18 h. Sizeexclusion chromatography (Superdex 200; 0.1 M Na₃PO₄, 0.15 M NaCl,pH=7.5) gave the purified conjugate which was diluted to an A₂₈₀=0.0375into Avidin Diluent with B5 Blocker (Ventana Medical Systems, Inc.,Tucson, Ariz.) and analyzed on tissue. A comparison of staining ofprostate specific antigen on prostate tissue using the HRP conjugate ofthis Example to an HRP conjugate prepared by DTT reduction of theimmunoglobulin as described in U.S. Provisional Patent Application, No.60/675,759 showed that the HRP conjugate of this Example exhibitedslightly less background than the DTT-prepared HRP conjugate, but alsoexhibited slightly less staining intensity.

Example 20 Multifunctional Hydrazide Thiol Linkers Derived From AminoAcids

In some embodiments, multifunctional hydrazide thiol linkers that can beused in the disclosed method are prepared from amino acids and aminoacid analogues according to schemes 4a, 4b, 4c and 4d above. In thisexample, synthetic routes to specific linkers are outlined in thefollowing schemes. In each of specific schemes 17a, 17b, 17c and 17d, anamino acid or amino acid analog (Sigma-Aldrich, St. Louis, Mo.) is firstreacted with N-Succinimidyl S-Acetylthioacetate (SATA; PierceBiotechnology, Rockford, Ill.) in the presence of triethylamine (TEA).In scheme 17 a, the product of this first reaction is reacted withhydrazine to provide a multifunctional hydrazide thiol linker having onehydrazide group and two thiol groups. In Scheme 17b,carbodiimide-meditated coupling with DCC is used to form an NHS activeester with the carboxylic acid functionality of the product of the firstreaction, followed by reaction with hydrazine to yield anothermultifunctional hydrazide thiol linker having one hydrazide group andtwo thiol groups. In Scheme 17c, as in 17b, NHS ester formation usingthe product of the first reaction is followed by reaction with hydrazineto yield a multifunctional hydrazide thiol linker having two hydrazidegroups and one thiol group. In Scheme 17d, the reaction with hydrazineyields a multifunctional hydrazide thiol linker having one hydrazidegroup, one thiol group and one hydroxyl group.

The products of Schemes 17a, 17b, 17c and 17d are, respectively,2-mercaptoacetamido-mercaptobutyric acid hydrazide (MAMBH),N,N′-(6-hydrazinyl-6-oxohexane-1,5-diyl)bis(2-mercaptoacetamide) (BTAL),N-(1,5-dihydrazinyl-1,5-dioxopentan-2-yl)-2-mercaptoacetamide (TAGD) andN-(1-hydrazinyl-4-hydroxy-1-oxobutan-2-yl)-2-mercaptoacetamide.

In a particular embodiment, MAMBH is synthesized as follows. FirstS-Acetylthioacetamide homocysteine is prepared by preparing a solutionof triethylamine (0.15 ml, 1.1 mmol) in acetonitrile (10 ml) to whichwas added homocysteine hydrochloride (150 mg, 1.0 mmol). The resultingslurry was stirred for 5 minutes before the addition ofS-acetylthioacetate (250 mg, 1.1 mmol). The reaction was stirred for 16h at ambient temperature and then concentrated in vacuo. Columnchromatography (SiO₂, 9:1 CH₂Cl₂/Et₂O) resulted in the isolation of theproduct as a colorless powder. Yield: 174 mg (75%): ¹H NMR (250 MHz,CDCl₃) δ 6.66 (bs, 1 H), 4.51-4.41 (p, J=6.7 Hz, 1 H), 3.63-3.50 (m, 2H), 3.36-3.18 (m, 2 H), 2.88-2.80 (m, 1 H), 2.38 (s, 3 H), 2.01-1.88 (m,1 H); ¹³C NMR (62.9 MHz, CDCl₃) δ 204.37, 195.38, 168.59, 59.51, 32.74,31.43, 30.18, 27.43; ESI-HRMS m/z 256.00693 (M+Na⁺, C₈H₁₁NNaO₃S₂ calcd256.00780). 2-Mercaptoacetamido-mercaptobutyric acid hydrazide (MAMBH)is then prepared by adding the S-acetylthioacetamide homocysteine (300mg, 1.3 mmol) to hydrazine monohydrate (10 ml). The resulting slurry wasstirred for 16 h at ambient temperature at which time the solutionbecomes homogeneous. The hydrazine was removed in vacuo and the crudeproduct was purified by reverse-phase flash chromatography (15% C₈ SiO₂,160:39:1 H₂O/MeOH/AcOH) to give the desired compound as a colorless oil.Yield: 207 mg (72%): ¹H NMR (250 MHz, CD₃OD) δ 4.52-4.46 (m, 1 H),3.23-3.21 (m, 2 H), 2.59-2.52 (m, 2 H), 2.10-2.01 (m, 2 H); ¹³C NMR(62.9 MHz, CD₃OD) δ 172.82, 172.43, 52.49, 37.72, 21.42, 20.49; ESI-HRMSm/z 246.03251 (M+Na⁺, C₆H₁₃N₃NaO₂S₂ calcd 246.03469).

Substitution of 6-Acetylthiohexanoic acid NHS ester for SATA in schemes17a, 17b and 17c yields the corresponding compounds TMBH, BTHL and THGDthat are shown below. 6-Acetylthiohexanoic acid NHS ester has thefollowing structure6-Acetylthiohexanoic acid NHS ester can be preparedfrom by carbodiimide mediated coupling of 6-Acetylthiohexanoic acid withN-Hydroxysuccinimide.

6-Acetylthiohexanoic acid NHS ester has the following structure:

and is prepared by carbodiimide mediated coupling of6-Acetylthiohexanoic acid with N-Hydroxysuccinimide (both available fromSigma-Aldrich, St. Louis, Mo.).

One of ordinary skill in the art also will recognize that PEG-basedS-acetyl-thiocarboxylic acid derivatives can be substituted for SATA inthe schemes above to provide multifunctional PEG-based linkers that beused in the disclosed method of conjugation. For example, PEG-basedmultifunctional hydrazide thiol linkers can be made by substituting amolecule of the following formula for SATA in schemes 17 a-d above:

wherein m=2 to 50. Compounds of this formula are commercially availablefrom Quanta Biodesign (Powell, Ohio), or can be prepared fromcorresponding carboxylic acids.

Example 21 Multifunctional PEG-based Hydrazide Thiol Linkers

In some embodiments, multifunctional PEG-based hydrazide thiol linkersthat can be used in the disclosed method are prepared according toschemes 5a, 5b and 5c above. In this example, synthetic routes tospecific linkers are outlined in the following schemes 18a, 18b and 18c.Specific protocols for the reactions also are presented. Unlessotherwise stated, reagents and solvents are conventional and can beobtained, for example, from Sigma-Aldrich (St. Louis, Mo.).

According to Scheme 18a, to a solution of 5.0 grams of Tris in 10 mlwater is added sodium carbonate (1.3 eq) followed by phenoxyacetylchloride (1.2 eq), and the reaction is allowed to stir on iceunder nitrogen for 16 hours. The precipitated amino protected product isthen washed three times with water and dried under vacuum. The purecompound resulting from this first reaction is obtained bychromatography on a C18 silica based column eluted withacetonitrile/H2O, 5-100% acetonitrile over 30 minutes. Mesylate groupsare then introduced by treating with triethylamine (4.0 eq) and methanesulfonyl chloride (5.0 eq) in DMF. The DMF is removed under vacuum, theresidue taken in dry DCM and the salts removed by filtration. Removal ofthe DCM under vacuum gives the crude mesylate 2 which is used withoutfurther purification. To a solution of the mesylate (0.3 eq) in dry DMFis added HO-dPEG_(4™-SATA ()1.0 eq; Quanta Biodesign, Powell, Ohio) andK₂CO₃ (1.5 eq) and the reaction allowed to stir under nitrogen for 16hours. The DMF is removed under vacuum, the residue taken in dry DCM andsalts are removed by filtration. Removal of the DCM under vacuumfollowed by silica gel chromatography gives the pegylated intermediate.The Pac protecting group is then removed by treating with Pd/C in amixture of EtAc/MeOH. The semicarbizide is then elaborated by treatingthe resulting intermediate first with carbonyl diimidizole (10 eq)followed by hydrazine (100 eq).

According to Scheme 18b, to a solution of alcohol (HO-PEG4-SATA, QuantaBiodesign, Powell, Ohio, 1.3 eq) in DCM is added 1.5 eq ofdiazo-diisopropyldicarboxylate followed by 1.8 eq of tributyl phosphine,and the reaction is stirred under dry nitrogen for 30 minutes. To theresulting suspension is then added 1.0 eq of the phenol in DCM and thereaction allowed to stir under dry nitrogen for 16 hrs. The phenol etherobtained after silica gel chromatography is then taken in neat hydrazineand the solution is microwaved to give the multi functional PEG-basedhydrazide thiol linker.

According to Scheme 18c, to a solution of 5.0 grams of Tris in 10 mlwater is added potassium carbonate (1.3 eq) followed by phenoxyacetylchloride (1.2 eq), and the reaction is allowed to stir on iceunder nitrogen for 16 hours. The precipitated amino protected product isthen washed three times with water and dried under vacuum. The alkynegroups are then introduced by treating with sodium hydride (3.0 eq) andpropargyl bromide (10 eq) in DMF to give the alkyne intermediate 1 aftersilica gel chromatography. To a solution of HO-PEG₄-SATA (QuantaBiodesign, Powell, Ohio) in DCM is added methane sulfonyl chloride (1.2eq) followed by triethyl amine (1.4 eq), and the reaction allowed tostir on ice under nitrogen for 16 hours. The triethyl amine salt is thenremoved by filtration and the mesylate product dried under vacuum. To asolution of the mesylated alcohol in DCM is added sodium azide (1.2 eq),and the reaction allowed to stir under nitrogen for 16 hours to affordthe azide intermediate 2 after silica gel chromatography. To a 1:1solution of t-butanol/water containing copper sulfate (0.2 eq) andsodium ascorbate (0.5 eq) is added one equivalent each of theintermediate alkyne 1 and the intermediate azide 2. The reaction thenstirred under nitrogen for sixteen hours to afford the intermediate withthe protected nitrogen after silica gel chromatography. The nitrogenprotecting group is then removed by treating with 10% Pd/C in a 1:1mixture of ethyl acetate and methanol and the free amine then obtainedby an acid-base work up. To a solution of the free amine in DCM is addedcarbonyl diimidizole (10 eq), and the reaction stirred under nitrogenfor four hours. The reaction is then concentrated under vacuum and theresidue taken into neat hydrazine. The solution is then microwave at100° C. for 1 hour to give the multifunctional PEG-based hydrazide thiollinker.

It will be readily apparent to one skilled in the art that the PEG-basedmolecules can be replaced with other SATA alcohols in these schemes toprovide additional multifunctional PEG-based hydrazide thiol linkers,and that PEG SATA alcohols of differing lengths can be substituted aswell.

Example 22 Synthesis of a Polyacrylamide Hydrazide Thiol Linker

In this example, a polymeric multivalent hydrazide thiol linker isprovided, which linker can be prepared according to Scheme 19 below.

In Scheme 19, x can be, for example, 100-500 and y can be, for example,10-50. L represents a thiolating reagent used to convert a portion ofthe hydrazide groups to thiol groups. Polyacrylamide hydrazide (PAH) canbe synthesized by the method provided in published U.S. PatentApplication No. 20050158770. Briefly, in a 100 mL round-bottom flaskfitted with a condenser, 20 mL polyacrylamide (1 mmol, 50% wt in water,Sigma-Aldrich, Milwaukee, Wis.) is mixed with 10 mL distilled (DI) waterand 20 mL hydrazine monohydrate (420 mmol, Sigma-Aldrich, Milwaukee,Wis.). The reaction is microwaved for 60 min. After cooling to roomtemperature, the reaction is precipitated with an equal volume ofmethanol, centrifuged and decanted. The residue is taken up in 50 mL DIwater and the precipitation repeated for a total of three times. Thefinal residue is dissolved in DI water and lyophilized to give a fine,white hygroscopic powder. In an appropriate solvent, the resulting PAHis reacted with a thiolating agent, such as a thiol-dPEG-NHS ester(Quanta Biodesign, Powell, Ohio) or Traut's reagent, to thiolate aportion (for example, approximately 50-75%) of available hydrazides (z=5to 40) and provide a polymeric multifunctional hydrazide thiol linkerthat can be used in the disclosed method. Additional thiolating reagentscan be found, for example, in Hermanson, “Bioconjugate Techniques,”Academic Press, San Diego, 1996, ISBN 0-12-342336-8, which isincorporated by reference herein.

It is possible to use/prepare the polyacrylamide hydrazide thiol linkerin one of two ways, either synthesize it first and use the disclosedconjugation method, or first react PAH with one molecule, thiolate thePAH, and then react the now thiolated first molecule with a secondmolecule.

Although the principles of the present invention have been describedwith reference to several illustrative embodiments, it should beapparent to those of ordinary skill in the art that the details of theembodiments may be modified without departing from such principles. Forexample, although the detailed description has focused onantibody-enzyme conjugates, the linkers and methods can be used toprepare any type of conjugate including conjugates of antibodies andother detectable labels such as nanoparticles (for example, metal andsemiconductor nanoparticles such as gold nanoparticles and quantum dots,respectively), fluorescent molecules, fluorogenic molecules, coloredmolecules, colorogenic molecules, and paramagnetic constructs (such aschelates of paramagnetic ions). Conjugates of antibodies for directedtherapies (for example, conjugates of antibodies with drug molecules,toxins and radioactive constructs such as chelates of radioactive metalions) also are contemplated. Although, specific examples provided showthe use of hydrazide thiol linkers having hydrazide and carbohydrazidegroups, any “hydrazide group” can be substituted for the hydrazide orcarbohydrazide groups shown in both the disclosed method and thedisclosed conjugate. Furthermore, it should be understood that while oneor more of a single hydrazide thiol linker can be used to form aconjugate, it is also possible to use multiple different hydrazide thiollinkers to form a conjugate. The disclosed conjugates can be used in anytype of assay where a specific binding molecule attached to a detectablelabel can be used, for example, in any type of immunoassay in additionto the illustrated immunohistochemical assays, or in any type of in situhybridization assay. Detection protocols can be performed manually or inan automated fashion. Furthermore, the disclosed linkers also can beused to modify surfaces for binding molecules to a substrate, and suchsurface modification reactions can be performed using the disclosedmethod. The present invention includes all modifications, variations,and equivalents thereof as fall within the scope and spirit of thefollowing claims.

We claim:
 1. A kit, comprising: a conjugate, comprising an antibody, adetectable label comprising an enzyme including about 17-25 maleimidegroups introduced into the enzyme by NHS-PEG-maleimide linkers, eachlinker being covalently bonded to an amino group of the enzyme, and aplurality of hydrazide thiol linkers, each hydrazide thiol linkercomprising one or more linking atoms positioned between a hydrazidegroup and a thiol group, wherein the hydrazide thiol linker is selectedfrom mercaptobutyric acid hydrazide (MBH), mercaptobutyric acidcarbohydrazide (MBCH), mercaptoacetamido-mercaptobutyric acid hydrazide(MAMBH), thiohexanamidomercaptobutyric acid hydrazide (THMBH),N,N′-(6-hydrazinyl-6-oxohexane-1,5-diyl)bis(2-mercaptoacetamide) (BTAL),bisthiohexanamidohydrazidolysine (BTHL),N-(1,5-dihydrazinyl-1,5-dioxopentan-2-yl)2-mercaptoacetamide (TAGD),thiohexamidoglutamic acid dihydrazide (THGD), a PEG-basedhydrazide-thiol linker, a multifunctional hydrazide thiol linker, aPEG-based multifunctional hydrazide thiol linker, a polyacrylamidehydrazide thiol linker, and combinations thereof, the hydrazide group ofeach hydrazide thiol linker being covalently bonded to an oxidizedglycosylated Fc portion of the antibody and the thiol group of one ofthe hydrazide thiol linkers being covalently bonded to the detectablelabel via one of the maleimide groups.
 2. The kit of claim 1, whereinthe hydrazide thiol linker comprises a PEG-based hydrazide thiol linker.3. The kit of claim 2, wherein the PEG-based hydrazide thiol linkercomprises a mercapto-dPEG-hydrazide linker.
 4. The kit of claim 1,wherein the hydrazide thiol linker comprises MBH or MBCH.
 5. The kit ofclaim 1, wherein the enzyme is alkaline phosphatase or horseradishperoxidase.
 6. The kit of claim 5, wherein the enzyme is alkalinephosphatase.
 7. The kit of claim 6, wherein the alkaline phosphatasecomprises cross-linked alkaline phosphatase.
 8. The kit of claim 1,wherein the antibody comprises an anti-hapten antibody.
 9. The kit ofclaim 1, wherein the antibody comprises an anti-antibody antibody.
 10. Amethod for detecting a molecule of interest in a cell or tissue sample,comprising: contacting the cell or the tissue sample with a primaryantibody specifically recognizing the molecule of interest in the cellor the tissue sample to provide the cell or the tissue sample bound withthe primary antibody contacting the cell or tissue sample with aconjugate comprising an antibody capable of specifically recognizing theprimary antibody, a detectable label comprising an enzyme includingabout 17-25 maleimide groups introduced into the enzyme byNHS-PEG-maleimide linkers, each linker being covalently bonded to anamino group of the enzyme, and a plurality of hydrazide thiol linkers,each hydrazide thiol linker comprising one or more linking atomspositioned between a hydrazide group and a thiol group, wherein thehydrazide thiol linker is selected from mercaptobutyric acid hydrazide(MBH), mercaptobutyric acid carbohydrazide (MBCH),mercaptoacetamido-mercaptobutyric acid hydrazide (MAMBH),thiohexanamidomercaptobutyric acid hydrazide (THMBH),N,N′-(6-hydrazinyl-6-oxohexane-1,5-diyl)bis(2-mercaptoacetamide) (BTAL),bisthiohexanamidohydrazidolysine (BTHL),N-(1,5-dihydrazinyl-1,5-dioxopentan-2-yl)2-mercaptoacetamide (TAGD),thiohexamidoglutamic acid dihydrazide (THGD), a PEG-basedhydrazide-thiol linker, a multifunctional hydrazide thiol linker, aPEG-based multifunctional hydrazide thiol linker, a polyacrylamidehydrazide thiol linker, and combinations thereof, the hydrazide group ofeach hydrazide thiol linker being covalently bonded to an oxidizedglycosylated Fc portion of the antibody and the thiol group of one ofthe hydrazide thiol linkers being covalently bonded to one of themaleimide groups on the enzyme; and detecting a signal generated by theconjugate that are bound to the primary antibody indicating the presenceof the molecule of interest in the cell or tissue sample.
 11. The methodof claim 10, wherein the hydrazide thiol linker comprises a PEG-basedhydrazide thiol linker.
 12. The method of claim 11, wherein thePEG-based hydrazide thiol linker comprises a mercapto-dPEG-hydrazidelinker.
 13. The method of claim 10, wherein the hydrazide thiol linkercomprises MBH or MBCH.
 14. The method of claim 10, wherein the enzyme isalkaline phosphatase or horseradish peroxidase.
 15. The method of claim14, wherein the enzyme is alkaline phosphatase.
 16. The method of claim15, wherein the alkaline phosphatase comprises cross-linked alkalinephosphatase.